Dermal filler compositions for fine line treatment

ABSTRACT

The present invention provides highly injectable, long-lasting hyaluronic acid-based hydrogel dermal filler compositions which are particularly advantageous for correction of fine lines in the face.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/914,274, filed on Mar. 7, 2018, which is a continuation of U.S.patent application Ser. No. 15/199,150, filed on Jun. 30, 2016, issuedas U.S. Pat. No. 9,950,092 on Apr. 24, 2018, which is a continuation ofU.S. patent application Ser. No. 13/615,193, filed on Sep. 13, 2012,issued as U.S. Pat. No. 9,408,797 on Aug. 9, 2016, which claims priorityto and the benefit of U.S. Provisional Patent Application No.61/534,780, filed on Sep. 14, 2011; U.S. patent application Ser. No.13/615,193 is a continuation-in-part of U.S. patent application Ser. No.13/593,313, filed on Aug. 23, 2012, issued as U.S. Pat. No. 9,393,263 onJul. 19, 2016, which is a continuation-in-part of U.S. patentapplication Ser. No. 13/486,754, filed on Jun. 1, 2012, issued as U.S.Pat. No. 9,149,422 on Oct. 6, 2015, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 61/493,309, filed onJun. 3, 2011, the entire disclosure of each of the foregoingapplications being incorporated herein in its entirety by this specificreference.

BACKGROUND

The present invention generally relates to dermal filler compositions,and more specifically relates to injectable dermal filler compositionsthat are effective for treatment of fine lines in skin.

Skin aging is a progressive phenomenon, occurs over time and can beaffected by lifestyle factors, such as alcohol consumption, tobacco andsun exposure. Aging of the facial skin can be characterized by atrophy,slackening, and fattening. Atrophy corresponds to a massive reduction ofthe thickness of skin tissue. Slackening of the subcutaneous tissuesleads to an excess of skin and ptosis and leads to the appearance ofdrooping cheeks and eye lids. Fattening refers to an increase in excessweight by swelling of the bottom of the face and neck. These changes aretypically associated with dryness, loss of elasticity, and roughtexture.

Hyaluronic acid (HA), also known as hyaluronan, is a non-sulfatedglycosaminoglycan that is distributed widely throughout the human bodyin connective, epithelial, and neural tissues. Hyaluronic acid isabundant in the different layers of the skin, where it has multiplefunctions such as, e.g., to ensure good hydration, to assist in theorganization of the extracellular matrix, to act as a filler material;and to participate in tissue repair mechanisms. However, with age, thequantity of hyaluronic acid, collagen, elastin, and other matrixpolymers present in the skin decreases. For example, repeated exposed toultra violet light, e.g., from the sun, causes dermal cells to bothdecrease their production of hyaluronan as well as increase the rate ofits degradation. This loss of materials results in various skinconditions such as, e.g., wrinkling, hollowness, loss of moisture andother undesirable conditions that contribute to the appearance of aging.

Injectable dermal fillers have been successfully used in treating theaging skin. The fillers can replace lost endogenous matrix polymers, orenhance/facilitate the function of existing matrix polymers, in order totreat these skin conditions. Hyaluronic acid-based dermal fillers havebecome increasingly popular, as hyaluronic acid is a substance naturallyfound throughout the human body. These fillers are generally welltolerated, nonpermanent, and a fairly low risk treatment for a widevariety of skin conditions.

Tyndall effect is an adverse event occurring in some patientsadministered with hyaluronic acid (HA)-based dermal fillers. Tyndalleffect is characterized by the appearance of a blue discoloration at theskin site where a dermal filler had been injected, which representsvisible hyaluronic acid seen through the translucent epidermis. Clinicalreports suggest that filler administration technique and skin propertiescan influence the manifestation of this adverse event. Fillers with highstiffness and elasticity are successfully used to correct areas on theface like nasolabial folds, cheeks, and chin without any fear of facialdiscoloration, as the materials are injected in the mid and deep dermisregions. However, when these filler materials are used to correctsuperficial, fine line wrinkles, for example, tear trough, glabellarlines periorbital lines, smile lines, or forehead, or mistakenly appliedtoo superficially in the upper regions of the dermis, a bluishdiscoloration of the skin is often observed. This phenomenon, which isthought to be the result of Tyndall effect, leaves a semi-permanentdiscoloration of the application sites, and sometimes disappears onlyafter the administration of hyaluronidase to degrade the fillermaterial. Consequently, Tyndall effect is more common in patientstreated for superficial fine line wrinkles. Prolonged manifestation ofTyndall effect, typically for several months as long as the gel lasts inthe skin, is a cause of major concern among patients.

HA-based dermal filler gels have been specifically formulated to treat“fine line ” wrinkles found around the tear trough, forehead,periobital, glabellar lines, etc. Commercially available HA “fine line”gels include Juvéderm Refine (G′ ˜67 Pa; G″/G′ ˜0.59, HA concentration18 mg/ml), Belotero Soft (G′ ˜28 Pa; G″/G′ ˜1.1, HA concentration 20mg/ml), Emervel Touch (G′ ˜56 Pa; G″/G′ ˜0.64, HA concentration 20mg/ml), Stylage S (G′ ˜192 Pa; G″/G′ ˜0.20, HA concentration 16 mg/ml),Teosyal First Lines (G′ 59 Pa; G″/G′ ˜0.53, HA concentration 20 mg/ml),Restylane Touch (G′ ˜489 Pa; G″/G′ ˜0.24, HA concentration 18 mg/ml).Though these gels are formulated to have low elastic moduli, forexample, by lightly crosslinking the linear HA chains with a smallamount of crosslinker and/or by reducing the final HA concentration ofthese gels, most of the commercially available “fine line” gels stillshow Tyndall effect in some patients, especially when when injectedsuperficially, for example, at a depth of less than about one mm.

Collagen-based gels can be employed in the treatment of superficialwrinkles and does not appear to cause Tyndall effect. Collagen basedgels are not highly favored as they have relatively poor duration in theskin and require pre-testing in individuals. Radiesse® (calciumhydroxylapatite) is a subdermal, injectable implant, whose principalcomponent is synthetic calcium hydroxylapatite, not hyaluronic acid.Unlike hyaluronic acid-based dermal fillers, calcium hydroxylapatite isnot transparent, and thus avoids the complication of the Tyndall effect.However, if placed too superficially, this filler can be seen as a whitesubstance immediately beneath the skin. Furthermore, compared tohyaluronic acid based fillers, Radiesse® requires a larger needle forinjection and is not typically recommended for use in the eye area.

It would be desirable to provide an injectable hyaluronic acid-baseddermal filler that does not exhibit the bluish discoloration attributedto Tyndall effect, even when injected superficially.

SUMMARY

The present invention describes compositions and formulation methods forpreparing HA-based dermal fillers that can be administered in the upperdermis without producing any bluish discoloration of the skin, or atleast no significant or noticeable bluish discoloration. Further, manyof the presently described filler gels of the invention have been foundto last significantly longer in vivo than current commercially availablegels. In some aspects of the invention, optically transparent dermalfillers useful for enhancing the appearance of the skin are providedwhich add volume and fullness, and reduce the appearance of even fineline wrinkles without “tyndalling”. The present compositions can beintroduced into fine lines in the skin, even in regions of thin skin andrather superficially, without causing the negative blue discolorationassociated with many conventional optically transparent dermal fillers.

More specifically, in one aspect of the present invention, long lasting,therapeutic dermal filler compositions are provided which generallycomprise a biocompatible polymer, for example, crosslinked hyaluronicacid component and an additive combined with the hyaluronic acidcomponent.

In one embodiment, the polymer is a polysaccharide, for example,hyaluronic acid. The hyaluronic acid includes a crosslinked componentand may further include a non-crosslinked component. The additive maycomprise a vitamin, for example, vitamin C, for example, a stabilizedform of vitamin C, or a vitamin C derivative, for example, L-ascorbicacid 2-glucoside (AA2G), ascobyl 3-aminopropyl phosphate (Vitagen) orsodium ascorbyl phosphate (AA2P).

In one aspect of the invention, the additive is a vitamin derivativewhich is covalently conjugated to the polymer by a suitable reactionprocess, for example, etherification, amidization or estherification.

In a broad aspect of the invention, a dermal filler composition isprovided, the composition comprising a hyaluronic acid componentcrosslinked with a crosslinking component, and an additive other thanthe crosslinking component. The hyaluronic acid component may bechemically conjugated to the additive. Further, the composition exhibitsreduced Tyndall effect when administered into a dermal region of apatient, relative to composition that is substantially identical exceptwithout the additive. The composition may further comprise otheradditives, for example, an anesthetic agent, such as lidocaine. In oneembodiment, the additive is a vitamin C derivative, for example, AA2G.In another embodiment, the additive is Vitagen.

In one embodiment, the hyaluronic acid component is chemicallyconjugated to the additive with degree of conjugation being betweenabout 3 mol % and about 40 mol, for example, between about 3 mol % andabout 10 mol %.

The composition may be substantially optically transparent. Thecompositions generally have a G′ value of between about 40 Pa and about100 Pa, for example, no greater than about 100 Pa and, for example, noless than about 40 Pa.

In another aspect of the invention, methods of treating fine lines inthe skin of a patient are provided. In one embodiment, the methodcomprises the steps of introducing, into skin of a patient, acomposition comprising a mixture of a hyaluronic acid component, acrosslinking component crosslinking the hyaluronic acid, and an additiveother than the crosslinking component, the composition beingsubstantially optically transparent, and wherein the compositionexhibits reduced Tyndall effect relative to composition that issubstantially identical except without the additive.

In another aspect of the invention, methods of improving aestheticappearance of a face are provided, the methods generally comprising thesteps of administering, to a dermal region of a patient, a substantiallyoptically transparent dermal filler composition that exhibits no orinsignificant Tyndall effect. The composition may be made by the stepsof providing hyaluronic acid, reacting a crosslinking agent with avitamin C derivative, adding the reacted crosslinking agent and vitaminC derivative to the hyaluronic acid to form a crosslinked hyaluronicacid composition including covalently conjugated vitamin C; andhomogenizing and neutralizing the crosslinked hyaluronic acidcomposition to obtain an injectable dermal filler composition. In someembodiments, the vitamin C derivative is AA2G. In other embodiments, thevitamin C derivative is Vitagen.

In yet another aspect of the invention, methods of reducing appearanceof fine lines in thin skin regions of a patient are provided, whereinthe method generally comprises administering to the patient a dermalfiller composition, at a depth of no greater than about 1 mm, asubstantially optically transparent hyaluronic acid based dermal fillercomposition including a vitamin C or a vitamin C derivative. In someembodiments, the composition is injected at a depth of a depth of nogreater than about 0.8 mm, no greater than about 0.6 mm, or no greaterthan about 0.4 mm.

In yet another aspect of the invention, a dermal filler composition isprovided which is substantially optically transparent, and generallycomprises a hyaluronic acid component crosslinked with a crosslinkingcomponent, and a vitamin C derivative covalently conjugated to thehyaluronic acid component. In an exemplary embodiment, the compositionand having a G′ value between about 40 Pa and about 100 Pa. Further, thecomposition may have a hyaluronic acid concentration of between about 18mg/g and about 30 mg/g. These compositions may be especially useful andeffective in treating fine lines or superficial creases in the skin, forexample, even in very thin skin, for example, skin having a thickness ofno greater than about 1 mm. In some embodiments, the compositions of theinvention last at least 3 months, at least 6 months or up to a yearafter being introduced into the skin.

These and other aspects and advantages of the present invention may bemore readily understood and appreciated with referenced to the followingdrawings and detailed description.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a representation of the structure of L-ascorbic acid2-glucoside (AA2G)

FIG. 2 is a representation of the structure of ascobyl 3-aminopropylphosphate (Vitagen).

FIG. 3 is a representation of the structure of sodium ascorbyl phosphate(AA2P).

FIG. 4 is a representation of the structure of 1,4-butanediol diglycidylether (BDDE).

FIG. 5 is a representation of the structure of pentaerythritol glycidalether (Star-PEG epoxide).

FIG. 6 is a representation of the structure of pentaerythritol(3-aminopropyl) ether (Star-PEG amine).

FIG. 7 is a Table showing conjugation degrees and G′ values for variousdermal filler compositions in accordance with the invention.

FIG. 8 is a Table showing conjugation degrees, HA concentration and G′values for HA-AA2G(BDDE) dermal filler compositions in accordance withthe invention.

FIG. 9 is a graphical representation of observed percent release of AsAfrom a solution of AA2G in PBS, in terms of time in minutes, for fourdifferent α-glucosidase concentrations.

FIG. 10 shows a representation of a release profile of free AsA fromconjugated dermal fillers in accordance with the invention (sustainedrelease) (AA2G conversion in mol % versus reaction time).

FIGS. 11A and 11B show additional release data for various dermalfillers in accordance with the invention.

FIG. 12 shows images of skin after superficial injection of HA baseddermal filler gels of the invention and some commercially available gelsfor fine line application.

FIG. 13 shows visual Tyndall scores of HA based dermal filler gels ofthe invention and certain commercially available gels for fine lineapplication.

FIG. 14 shows % of blue light remitted from skin of HA based dermalfiller gels of the invention and some commercially available gels forfine line application.

FIG. 15 shows overall % of gel remaining after 1 week implantation of HAbased dermal filler gels of the invention and some commerciallyavailable gels for fine line application.

FIG. 16 shows overall % of gel remaining at Week 0 , Week 12, Week 24and Week 40 of implanted HA based dermal filler gels of the inventionand some commercially available gels for fine line application.

DETAILED DESCRIPTION

In one aspect of the invention, dermal filler compositions are provided,the compositions generally comprising a biocompatible polymer, forexample, a polysaccharide such as a crosslinked hyaluronic acid, and avitamin C derivative covalently conjugated to the polymer. Thecomposition is provides sustained release of the vitamin C for skinneocollagenesis as well as other therapeutic or cosmetic benefits. Whenintroduced into the skin, for example intradermally, the compositionreacts with endogeneous enzymes in the body, and over time, bioactivevitamin C is generated in vivo, via enzymatic cleavages. As vitamin C isreleased from the composition over a period of weeks or months, itsattendant benefits are made available to the body.

The polymer may be selected from the group of polymers consisting ofproteins, peptides and polypeptides, polylysine, collagens,pro-collagens, elastins, and laminins.

The polymer may be selected from the group of polymers consisting ofsynthetic polymers with hydroxyl, amine, and carboxyl functional groups:poly(vinyl alcohol), polyethylene glycol, polyvinlyl amine,polyallylamine, deacetylated polyacrylamide, polyacrylic acid, andpolymethacrylic acid. The polymer may be selected from the group ofpolymers consisting of dentric or branched polymers, including dentricpolyols and dentric polyamines. The polymer may be selected from thegroup of polymers consisting of solid surface with hydroxyl, amine, andcarboxyl functional groups.

The polymer may be a polysaccharide, for example, selected from thegroup of polysaccharides including starch and its derivatives; dextranand its derivatives, cellulose and its derivatives; chitin and chitosanand alginate and its derivatives.

In an exemplary embodiment of the invention, the polymer isglycosaminoglycan. The hydrogel composition disclosed herein can furthercomprise two or more different glycosaminoglycan polymers. As usedherein, the term “glycosaminoglycan” is synonymous with “GAG” and“mucopolysaccharide” and refers to long unbranched polysaccharidesconsisting of a repeating disaccharide units. The repeating unitconsists of a hexose (six-carbon sugar) or a hexuronic acid, linked to ahexosamine (six-carbon sugar containing nitrogen) and pharmaceuticallyacceptable salts thereof. Members of the GAG family vary in the type ofhexosamine, hexose or hexuronic acid unit they contain, such as, e.g.,glucuronic acid, iduronic acid, galactose, galactosamine, glucosamine)and may also vary in the geometry of the glycosidic linkage. Anyglycosaminoglycan polymer is useful in the hydrogel compositionsdisclosed herein with the proviso that the glycosaminoglycan polymerimproves a condition of the skin. Non-limiting examples ofglycosaminoglycans include chondroitin sulfate, dermatan sulfate,keratan sulfate, hyaluronan. Non-limiting examples of an acceptable saltof a glycosaminoglycans includes sodium salts, potassium salts,magnesium salts, calcium salts, and combinations thereof.Glycosaminoglycan and their resulting polymers useful in the hydrogelcompositions and methods disclosed herein are described in, e.g., Pironand Tholin, Polysaccharide Crosslinking, Hydrogel Preparation, ResultingPolysaccharides(s) and Hydrogel(s), uses Thereof, U.S. PatentPublication 2003/0148995; Lebreton, Cross-Linking of Low and HighMolecular Weight Polysaccharides Preparation of Injectable MonophaseHydrogels; Lebreton, Viscoelastic Solutions Containing SodiumHyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses,U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-BasedGels Including Lidocaine, U.S. Patent Publication 2010/0028438; andPolysaccharides and Hydrogels thus Obtained, U.S. Patent Publication2006/0194758; and Di Napoli, Composition and Method for Intradermal SoftTissue Augmentation, International Patent Publication WO 2004/073759,each of which is hereby incorporated by reference in its entirety. GAGsuseful in the hydrogel compositions and methods disclosed herein arecommercially available, such as, e.g., hyaluronan-based dermal fillersJUVEDERM®, JUVEDERM® 30, JUVEDERM® Ultra, JUVEDERM® Ultra Plus,JUVEDERM® Ultra XC, and JUVEDERM® Ultra Plus XC (Allergan Inc, Irvine,Calif.). Table 1 lists representative GAGs.

TABLE 1 Examples of GAGs Glycosidic Hexuronic linkage Name acid/HexoseHexosamine geometry Unique features Chondroitin GlcUA or GalNAc or-4GlcUAβ1- Most prevalent GAG sulfate GlcUA(2S) GalNAc(4S) or 3GalNAcβ1-GalNAc(6S) or GalNAc(4S,6S) Dermatan GlcUA or GalNAc or -4IdoUAβ1-Distinguished from sulfate IdoUA or GalNAc(4S) or 3GalNAcβ1- chondroitinsulfate by the IdoUA(2S) GalNAc(6S) or presence of iduronic acid,GalNAc(4S,6S) although some hexuronic acid monosaccharides may beglucuronic acid. Keratan Gal or GlcNAc or -3Gal(6S)β1- Keratan sulfatetype II may sulfate Gal(6S) GlcNAc(6S) 4GlcNAc(6S)β1- be fucosylated.Heparin GlcUA or GlcNAc or -4IdoUA(2S)α1- Highest negative chargeIdoUA(2S) GlcNS or 4GlcNS(6S)α1- density of any known GlcNAc(6S) orbiological molecule GlcNS(6S) Heparan GlcUA or GlcNAc or -4GlcUAβ1-Highly similar in structure sulfate IdoUA or GlcNS or 4GlcNAcα1- toheparin, however IdoUA(2S) GlcNAc(6S) or heparan sulfates GlcNS(6S)disaccharide units are organized into distinct sulfated and non-sulfateddomains. Hyaluronan GlcUA GlcNAc -4GlcUAβ1- The only GAG that is3GlcNAcβ1- exclusively non-sulfated GlcUA = β-D-glucuronic acidGlcUA(2S) = 2-O-sulfo-β-D-glucuronic acid IdoUA = α-L-iduronic acidIdoUA(2S) = 2-O-sulfo-α-L-iduronic acid Gal = β-D-galactose Gal(6S) =6-O-sulfo-β-D-galactose GalNAc = β-D-N-acetylgalactosamine GalNAc(4S) =β-D-N-acetylgalactosamine-4-O-sulfate GalNAc(6S) =β-D-N-acetylgalactosamine-6-O-sulfate GalNAc(4S,6S) =β-D-N-acetylgalactosamine-4-O, 6-O-sulfate GlcNAc =α-D-N-acetylglucosamine GlcNS = α-D-N-sulfoglucosamine GlcNS(6S) =α-D-N-sulfoglucosamine-6-O-sulfate

Aspects of the present invention provide, in part, a hydrogelcomposition comprising a chondroitin sulfate polymer. As used herein,the term “chondroitin sulfate polymer” refers to an unbranched, sulfatedpolymer of variable length comprising disaccharides of two alternatingmonosaccharides of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine(GalNAc) and pharmaceutically acceptable salts thereof. A chondroitinsulfate polymer may also include D-glucuronic acid residues that areepimerized into L-iduronic acid (IdoA), in which case the resultingdisaccharide is referred to as dermatan sulfate. A chondroitin sulfatepolymer can have a chain of over 100 individual sugars, each of whichcan be sulfated in variable positions and quantities. Chondroitinsulfate polymers are an important structural component of cartilage andprovide much of its resistance to compression. Any chondroitin sulfatepolymer is useful in the compositions disclosed herein with the provisothat the chondroitin sulfate polymer improves a condition of the skin.Non-limiting examples of pharmaceutically acceptable salts ofchondroitin sulfate include sodium chondroitin sulfate, potassiumchondroitin sulfate, magnesium chondroitin sulfate, calcium chondroitinsulfate, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a keratan sulfate polymer. As used herein, theterm “keratan sulfate polymer” refers to a polymer of variable lengthcomprising disaccharide units, which themselves include β-D-galactoseand N-acetyl-D-galactosamine (GalNAc) and pharmaceutically acceptablesalts thereof. Disaccharides within the repeating region of keratansulfate may be fucosylated and N-Acetylneuraminic acid caps the end ofthe chains. Any keratan sulfate polymer is useful in the compositionsdisclosed herein with the proviso that the keratan sulfate polymerimproves a condition of the skin. Non-limiting examples ofpharmaceutically acceptable salts of keratan sulfate include sodiumkeratan sulfate, potassium keratan sulfate, magnesium keratan sulfate,calcium keratan sulfate, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a hyaluronan polymer. As used herein, the term“hyaluronic acid polymer” is synonymous with “HA polymer”, “hyaluronicacid polymer”, and “hyaluronate polymer” refers to an anionic,non-sulfated glycosaminoglycan polymer comprising disaccharide units,which themselves include D-glucuronic acid and D-N-acetylglucosaminemonomers, linked together via alternating β-1,4 and β-1,3 glycosidicbonds and pharmaceutically acceptable salts thereof. Hyaluronan polymerscan be purified from animal and non-animal sources. Polymers ofhyaluronan can range in size from about 5,000 Da to about 20,000,000 Da.Any hyaluronan polymer is useful in the compositions disclosed hereinwith the proviso that the hyaluronan improves a condition of the skin.Non-limiting examples of pharmaceutically acceptable salts of hyaluronaninclude sodium hyaluronan, potassium hyaluronan, magnesium hyaluronan,calcium hyaluronan, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a crosslinked glycosaminoglycan polymer. As usedherein, the term “crosslinked” refers to the intermolecular bondsjoining the individual polymer molecules, or monomer chains, into a morestable structure like a gel. As such, a crosslinked glycosaminoglycanpolymer has at least one intermolecular bond joining at least oneindividual polymer molecule to another one. The crosslinking ofglycosaminoglycan polymers typically result in the formation of ahydrogel. Such hydrogels have high viscosity and require considerableforce to extrude through a fine needle. Glycosaminoglycan polymersdisclosed herein may be crosslinked using dialdehydes and disulfidescrosslinking agents including, without limitation, multifunctionalPEG-based crosslinking agents, divinyl sulfones, diglycidyl ethers, andbis-epoxides, biscarbodiimide. Non-limiting examples of hyaluronancrosslinking agents include multifunctional PEG-based crosslinkingagents like pentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone(DVS), 1,4-butanediol diglycidyl ether (BDDE),1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),(phenylenebis-(ethyl)-carbodiimide and 1,6 hexamethylenebis(ethylcarbodiimide), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (NMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, lysine, lysine methylester, orcombinations thereof. Other useful cross-linking agents are disclosed inStroumpoulis and Tezel, Tunably Crosslinked Polysaccharide Compositions,U.S. patent application Ser. No. 12/910,466, filed Oct. 22, 2010, whichis incorporated by reference in its entirety. Non-limiting examples ofmethods of crosslinking glycosaminoglycan polymers are described in,e.g., Glycosaminoglycan polymers useful in the compositions and methodsdisclosed herein are described in, e.g., Piron and Tholin,Polysaccharide Crosslinking, Hydrogel Preparation, ResultingPolysaccharides(s) and Hydrogel(s), uses Thereof, U.S. PatentPublication 2003/0148995; Lebreton, Cross-Linking of Low and HighMolecular Weight Polysaccharides Preparation of Injectable MonophaseHydrogels; Lebreton, Viscoelastic Solutions Containing SodiumHyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses,U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-BasedGels Including Lidocaine, U.S. Patent Publication 2010/0028438; andPolysaccharides and Hydrogels thus Obtained, U.S. Patent Publication2006/0194758; and Di Napoli, Composition and Method for Intradermal SoftTissue Augmentation, International Patent Publication WO 2004/073759,each of which is hereby incorporated by reference in its entirety.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a crosslinked glycosaminoglycan polymer having adegree of crosslinking. As used herein, the term “degree ofcrosslinking” refers to the percentage of glycosaminoglycan polymermonomeric units, such as, e.g., the disaccharide monomer units ofhyaluronan that are bound to a cross-linking agent. The degree ofcrosslinking is expressed as the percent weight ratio of thecrosslinking agent to glycosaminoglycan. The degree of crosslinking incertain advantageous embodiment of the invention is between about 3% andabout 12%, for example, between about 5% and about 10%.

In an embodiment, a hydrogel composition comprises a crosslinkedglycosaminoglycan polymer, for example, crosslinked hyaluronic acid,wherein the crosslinked glycosaminoglycan polymer is present in thecomposition at a concentration of, for example, between about 18 mg/gand about 30 mg/g. In some embodiments, the compositions have a totalhyaluronic acid concentration of about 24 mg/g or about 25 mg/g.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising hyaluronan polymers of low molecular weight,hyaluronan polymers of high molecular weight, or hyaluronan polymers ofboth low and high molecular weight. As used herein, the term “highmolecular weight” when referring to “hyaluronan” refers to hyaluronanpolymers having a mean molecular weight of 1,000,000 Da or greater.Non-limiting examples of a high molecular weight hyaluronan polymersinclude hyaluronan polymers about 1,500,000 Da, about 2,000,000 Da,about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Da, about4,000,000 Da, about 4,500,000 Da, and about 5,000,000 Da. As usedherein, the term “low molecular weight” when referring to “hyaluronan”refers to hyaluronan polymers having a mean molecular weight of lessthan 1,000,000 Da. Non-limiting examples of a low molecular weighthyaluronan polymers include hyaluronan polymers of about 200,000 Da,about 300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da,about 700,000 Da, of about 800,000 Da, and about 900,000 Da.

In an embodiment, a composition comprises crosslinked hyaluronanpolymers of low molecular weight. In aspects of this embodiment, acomposition comprises crosslinked hyaluronan polymers having a meanmolecular weight of, e.g., about 100,000 Da, about 200,000 Da, about300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da, about700,000 Da, about 800,000 Da, or about 900,000 Da. In yet other aspectsof this embodiment, a composition comprises crosslinked hyaluronanpolymers having a mean molecular weight of, e.g., at most 100,000 Da, atmost 200,000 Da, at most 300,000 Da, at most 400,000 Da, at most 500,000Da, at most 600,000 Da, at most 700,000 Da, at most 800,000 Da, at most900,000 Da, or at most 950,000 Da. In still other aspects of thisembodiment, a composition comprises crosslinked hyaluronan polymershaving a mean molecular weight of, e.g., about 100,000 Da to about500,000 Da, about 200,000 Da to about 500,000 Da, about 300,000 Da toabout 500,000 Da, about 400,000 Da to about 500,000 Da, about 500,000 Dato about 950,000 Da, about 600,000 Da to about 950,000 Da, about 700,000Da to about 950,000 Da, about 800,000 Da to about 950,000 Da, about300,000 Da to about 600,000 Da, about 300,000 Da to about 700,000 Da,about 300,000 Da to about 800,000 Da, or about 400,000 Da to about700,000 Da.

In another embodiment, a composition comprises crosslinked hyaluronanpolymers of high molecular weight. In aspects of this embodiment, acomposition comprises a crosslinked hyaluronan polymers having a meanmolecular weight of, e.g., about 1,000,000 Da, about 1,500,000 Da, about2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000Da, about 4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da. Inyet other aspects of this embodiment, a composition comprises acrosslinked hyaluronan polymers having a mean molecular weight of, e.g.,at least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da, atleast 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000 Da, atleast 4,000,000 Da, at least 4,500,000 Da, or at least 5,000,000 Da. Instill other aspects of this embodiment, a composition comprises acrosslinked hyaluronan polymers having a mean molecular weight of, e.g.,about 1,000,000 Da to about 5,000,000 Da, about 1,500,000 Da to about5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da, about 2,500,000Da to about 5,000,000 Da, about 2,000,000 Da to about 3,000,000 Da,about 2,500,000 Da to about 3,000,000 Da.

In yet another embodiment, a composition comprises a crosslinkedhyaluronan polymers where the crosslinked hyaluronan polymers comprise acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers, in various ratios. In aspects ofthis embodiment, a composition comprises a crosslinked hyaluronanpolymers where the crosslinked hyaluronan polymers comprises acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers in a ratio of about 20:1, about15:1, about 10:1, about 5:1, about 1:1, about 1:5 about 1:10, about1:15, or about 1:20.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising an uncrosslinked glycosaminoglycan polymer. Asused herein, the term “uncrosslinked” refers to a lack of intermolecularbonds joining the individual glycosaminoglycan polymer molecules, ormonomer chains. As such, an uncrosslinked glycosaminoglycan polymer isnot linked to any other glycosaminoglycan polymer by an intermolecularbond. In aspects of this embodiment, a composition comprises anuncrosslinked chondroitin sulfate polymer, an uncrosslinked dermatansulfate polymer, an uncrosslinked keratan sulfate polymer, anuncrosslinked heparan polymer, an uncrosslinked heparan sulfate polymer,or an uncrosslinked hyaluronan polymer. Uncrosslinked glycosaminoglycanpolymers are water soluble and generally remain fluid in nature. Assuch, uncross-linked glycosaminoglycan polymers are often mixed with aglycosaminoglycan polymer-based hydrogel composition as a lubricant tofacilitate the extrusion process of the composition through a fineneedle.

In an embodiment, a composition comprises an uncrosslinkedglycosaminoglycan polymer where the uncrosslinked glycosaminoglycanpolymer is present at a concentration of, e.g., about 2 mg/g, about 3mg/g, about 4 mg/g, about 5 mg/g, about 6 mg/g, about 7 mg/g, about 8mg/g, about 9 mg/g, about 10 mg/g, about 11 mg/g, about 12 mg/g, about13 mg/g, about 13.5 mg/g, about 14 mg/g, about 15 mg/g, about 16 mg/g,about 17 mg/g, about 18 mg/g, about 19 mg/g, about 20 mg/g, about 40mg/g, or about 60 mg/g. In other aspects of this embodiment, acomposition comprises an uncrosslinked glycosaminoglycan where theuncrosslinked glycosaminoglycan is present at a concentration of, e.g.,at least 1 mg/g, at least 2 mg/g, at least 3 mg/g, at least 4 mg/g, atleast 5 mg/g, at least 10 mg/g, at least 15 mg/g, at least 20 mg/g, atleast 25 mg/g at least 35 mg/g, or at least 40 mg/g. In yet otheraspects of this embodiment, a composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., at most 1 mg/g, at most 2 mg/g, at most 3mg/g, at most 4 mg/g, at most 5 mg/g, at most 10 mg/g, at most 15 mg/g,at most 20 mg/g, or at most 25 mg/g. In still other aspects of thisembodiment, a composition comprises an uncrosslinked glycosaminoglycanwhere the uncrosslinked glycosaminoglycan is present at a concentrationof, e.g., about 1 mg/g to about 60 mg/g, about 10 mg/g to about 40 mg/g,about 7.5 mg/g to about 19.5 mg/g, about 8.5 mg/g to about 18.5 mg/g,about 9.5 mg/g to about 17.5 mg/g, about 10.5 mg/g to about 16.5 mg/g,about 11.5 mg/g to about 15.5 mg/g, or about 12.5 mg/g to about 14.5mg/g.

Aspects of the present specification provide, in part, a hydrogelcomposition that is essentially free of a crosslinked glycosaminoglycanpolymer. As used herein, the term “essentially free” (or “consistingessentially of”) refers to a composition where only trace amounts ofcross-linked matrix polymers can be detected. In an aspect of thisembodiment, a composition comprises a chondroitin sulfate that isessentially free of a crosslinked chondroitin sulfate polymer, adermatan sulfate essentially free of a crosslinked dermatan sulfatepolymer, a keratan sulfate essentially free of a crosslinked keratansulfate polymer, a heparan essentially free of a crosslinked heparanpolymer, a heparan sulfate essentially free of a crosslinked heparansulfate polymer, or a hyaluronan sulfate essentially free of acrosslinked hyaluronan polymer.

Aspects of the present specification provide, in part, a hydrogelcomposition that is entirely free of a crosslinked glycosaminoglycanpolymer. As used herein, the term “entirely free” refers to acomposition that within the detection range of the instrument or processbeing used, crosslinked glycosaminoglycan polymers cannot be detected orits presence cannot be confirmed. In an aspect of this embodiment, acomposition comprises a chondroitin sulfate that is entirely free of acrosslinked chondroitin sulfate polymer, a dermatan sulfate entirelyfree of a crosslinked dermatan sulfate polymer, a keratan sulfateentirely free of a crosslinked keratan sulfate polymer, a heparanentirely free of a crosslinked heparan polymer, a heparan sulfateentirely free of a crosslinked heparan sulfate polymer, or a hyaluronansulfate entirely free of a crosslinked hyaluronan polymer.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a ratio of crosslinked glycosaminoglycan polymerand uncrosslinked glycosaminoglycan polymer. This ratio of crosslinkedand uncrosslinked glycosaminoglycan polymer is also known as thegel:fluid ratio. Any gel:fluid ratio is useful in making thecompositions disclosed herein with the proviso that such ratio producesa composition disclosed herein that improves a skin condition asdisclosed herein. Non-limiting examples of gel:fluid ratios incompositions of the present invention include 100:0, 98:2, 90:10, 75:25,70:30, 60:40, 50:50, 40:60, 30:70, 25:75, 10:90; 2:98, and 0:100.

In aspects of this embodiment, a composition comprises a crosslinkedglycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymerwhere the gel:fluid ratio is, e.g., about 0:100, about 1:99, about 2:98,about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92,about 9:91, or about 10:90. In other aspects of this embodiment, acomposition comprises a crosslinked glycosaminoglycan polymer and anuncrosslinked glycosaminoglycan polymer where the gel:fluid ratio is,e.g., at most 1:99, at most 2:98, at most 3:97, at most 4:96, at most5:95, at most 6:94, at most 7:93, at most 8:92, at most 9:91, or at most10:90. In yet other aspects of this embodiment, a composition comprisesa crosslinked glycosaminoglycan polymer and an uncrosslinkedglycosaminoglycan polymer where the gel:fluid ratio is, e.g., about0:100 to about 3:97, about 0:100 to about 5:95, or about 0:100 to about10:90.

In other aspects of this embodiment, a composition comprises acrosslinked glycosaminoglycan polymer and an uncrosslinkedglycosaminoglycan polymer where the gel:fluid ratio is, e.g., about15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60,about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5,about 98:2, or about 100:0. In yet other aspects of this embodiment, acomposition comprises a crosslinked glycosaminoglycan polymer and anuncrosslinked glycosaminoglycan polymer where the gel:fluid ratio is,e.g., at most 15:85, at most 20:80, at most 25:75, at most 30:70, atmost 35:65, at most 40:60, at most 45:55, at most 50:50, at most 55:45,at most 60:40, at most 65:35, at most 70:30, at most 75:25, at most80:20, at most 85:15, at most 90:10, at most 95:5, at most 98:2, or atmost 100:0. In still other aspects of this embodiment, a compositioncomprises a crosslinked glycosaminoglycan polymer and an uncrosslinkedglycosaminoglycan polymer where the gel:fluid ratio is, e.g., about10:90 to about 70:30, about 15:85 to about 70:30, about 10:90 to about55:45, about 80:20 to about 95:5, about 90:10 to about 100:0, about75:25 to about 100:0, or about 60:40 to about 100:0.

A hydrogel composition disclosed herein may further comprise anotheragent or combination of agents that provide a beneficial effect when thecomposition is administered to an individual. Such beneficial agentsinclude, without limitation, an antioxidant, an anti-itch agent, ananti-cellulite agent, an anti-scarring agent, an anti-inflammatoryagent, an anesthetic agent, an anti-irritant agent, a vasoconstrictor, avasodilator, an anti-hemorrhagic agent like a hemostatic agent oranti-fibrinolytic agent, a desquamating agent, a tensioning agent, ananti-acne agent, a pigmentation agent, an anti-pigmentation agent, or amoisturizing agent.

For purposes of the present specification, unless otherwise stated, “%”in a formulation is defined as weight by weight (i.e., w/w) percentage.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that may optionally comprise an anestheticagent. An anesthetic agent is preferably a local anesthetic agent, i.e.,an anesthetic agent that causes a reversible local anesthesia and a lossof nociception, such as, e.g., aminoamide local anesthetics andaminoester local anesthetics. The amount of an anesthetic agent includedin a composition disclosed herein is an amount effective to mitigatepain experienced by an individual upon administration of thecomposition. As such, the amount of an anesthetic agent included in acomposition disclosed in the present specification is between about 0.1%to about 5% by weight of the total composition. Non-limiting examples ofanesthetic agents include lidocaine, ambucaine, amolanone, amylocaine,benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine,butacaine, butamben, butanilicaine, butethamine, butoxycaine,carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine,dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine,ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine,euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine,isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine,mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine,naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine,phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine,prilocaine, procaine, propanocaine, proparacaine, propipocaine,propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol,tetracaine, tolycaine, trimecaine, zolamine, combinations thereof, andsalts thereof. Non-limiting examples of aminoester local anestheticsinclude procaine, chloroprocaine, cocaine, cyclomethycaine, cimethocaine(larocaine), propoxycaine, procaine (novocaine), proparacaine,tetracaine (amethocaine). Non-limiting examples of aminoamide localanesthetics include articaine, bupivacaine, cinchocaine (dibucaine),etidocaine, levobupivacaine, lidocaine (lignocaine), mepivacaine,piperocaine, prilocaine, ropivacaine, and trimecaine. A compositiondisclosed herein may comprise a single anesthetic agent or a pluralityof anesthetic agents. A non-limiting example of a combination localanesthetic is lidocaine/prilocaine (EMLA).

Thus in an embodiment, a composition disclosed herein comprises ananesthetic agent and salts thereof. In aspects of this embodiment, acomposition disclosed herein comprises an aminoamide local anestheticand salts thereof or an aminoester local anesthetic and salts thereof.In other aspects of this embodiment, a composition disclosed hereincomprises procaine, chloroprocaine, cocaine, cyclomethycaine,cimethocaine, propoxycaine, procaine, proparacaine, tetracaine, or saltsthereof, or any combination thereof. In yet other aspects of thisembodiment, a composition disclosed herein comprises articaine,bupivacaine, cinchocaine, etidocaine, levobupivacaine, lidocaine,mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, or saltsthereof, or any combination thereof. In still other aspects of thisembodiment, a composition disclosed herein comprises alidocaine/prilocaine combination.

In other aspects of this embodiment, a composition disclosed hereincomprises an anesthetic agent in an amount of, e.g., about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8% about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10% byweight of the total composition. In yet other aspects, a compositiondisclosed herein comprises an anesthetic agent in an amount of, e.g., atleast 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%,at least 0.6%, at least 0.7%, at least 0.8% at least 0.9%, at least1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, atleast 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, or at least 10%by weight of the total composition. In still other aspects, acomposition disclosed herein comprises an anesthetic agent in an amountof, e.g., at most 0.1%, at most 0.2%, at most 0.3%, at most 0.4%, atmost 0.5%, at most 0.6%, at most 0.7%, at most 0.8% at most 0.9%, atmost 1.0%, at most 2.0%, at most 3.0%, at most 4.0%, at most 5.0%, atmost 6.0%, at most 7.0%, at most 8.0%, at most 9.0%, or at most 10% byweight of the total composition. In further aspects, a compositiondisclosed herein comprises an anesthetic agent in an amount of, e.g.,about 0.1% to about 0.5%, about 0.1% to about 1.0%, about 0.1% to about2.0%, about 0.1% to about 3.0%, about 0.1% to about 4.0%, about 0.1% toabout 5.0%, about 0.2% to about 0.9%, about 0.2% to about 1.0%, about0.2% to about 2.0%, about 0.5% to about 1.0%, or about 0.5% to about2.0% by weight of the total composition.

In another embodiment, a composition disclosed herein does not comprisean anesthetic agent.

In one aspect of the present invention, an injectable dermal filler isprovided which comprises a polymer, for example, a glycosaminoglycanpolymer, for example a hyaluronic acid polymer, for example, ahyaluronic acid at least a portion of which is crosslinked, and anadditive or beneficial agent combined with the polymer.

The beneficial agent combined with the polymer may comprise a vitamin,for example, vitamin C. Non-limiting examples of suitable forms ofvitamin C include ascorbic acid and sodium, potassium, and calcium saltsof ascorbic acid, fat-soluble esters of ascorbic acid with long-chainfatty acids (ascorbyl palm itate or ascorbyl stearate), magnesiumascorbyl phosphate (MAP), sodium ascorbyl phosphate (SAP), and ascorbicacid 2-glucoside (AA2G™), sodium ascorbyl phosphate (AA2P), disodiumascorbyl sulfate, and ascobyl 3-aminopropyl phosphate (Vitagen).

In an especially advantageous embodiment, the beneficial agent iscovalently conjugated to the polymer. For example, the beneficial agentmay be a vitamin C, or a vitamin C derivative, which is covalentlyconjugated to the polymer and is present in the compositions in anamount between about 0.04% to about 5.0% by weight of the totalcomposition, for example, between about 0.1% to about 4.0% by weight ofthe total composition, for example, between about 0.2% to about 2.0% byweight of the total composition. In one embodiment, the amount ofvitamin C included in a composition disclosed herein is between about0.3% to about 1.2% by weight of the total composition.

Preferably, the vitamin C covalently conjugated to the polymer, includesat least one of ascorbic acid, L-ascorbic acid, L-ascorbic acid2-sulfate (AA-2S) and L-ascorbic acid 2-phosphate (AA-2P), ascorbic acid2-O-glucoside (AA-2G), 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbicacids (6-Acyl-AA-2G), (ascobyl 3-aminopropyl phosphate, Ascorbylpalmitate), derivatives and combinations thereof. A compositiondisclosed herein may comprise a single vitamin C agent or a plurality ofvitamin C agents.

In another embodiment of the invention, a dermal filler is providedwherein the hyaluronic acid is crosslinked with BDDE. In thisembodiment, the degree of conjugation may be between about 3 mol % andabout 10 mol %, to about 15 mol % to about 40 mol %.

In some embodiments, the dermal fillers have a sustainedbioavailability. For example, dermal fillers are provided which, whenintroduced into the skin of a human being, are effective to releaseascorbic acid or other vitamin into the human being for at least about 1months and up to about 20 months or more.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits a complex modulus, an elasticmodulus, a viscous modulus and/or a tan δ. The compositions as disclosedherein are viscoelastic in that the composition has an elastic component(solid-like such as, e.g., crosslinked glycosaminoglycan polymers) and aviscous component (liquid-like such as, e.g., uncrosslinkedglycosaminoglycan polymers or a carrier phase) when a force is applied(stress, deformation). The rheological attribute that described thisproperty is the complex modulus (G*), which defines a composition'stotal resistance to deformation. The complex modulus is a complex numberwith a real and imaginary part: G*=G′+iG″. The absolute value of G* isAbs(G*)=Sqrt(G′²+G″²). The complex modulus can be defined as the sum ofthe elastic modulus (G′) and the viscous modulus (G″). Falcone, et al.,Temporary Polysaccharide Dermal Fillers: A Model for Persistence Basedon Physical Properties, Dermatol Surg. 35(8): 1238-1243 (2009); Tezel,supra, 2008; Kablik, supra, 2009; Beasley, supra, 2009; each of which ishereby incorporated by reference in its entirety.

Elastic modulus, or modulus of elasticity, refers to the ability of ahydrogel material to resists deformation, or, conversely, an object'stendency to be non-permanently deformed when a force is applied to it.Elastic modulus characterizes the firmness of a composition and is alsoknown as the storage modulus because it describes the storage of energyfrom the motion of the composition. The elastic modulus describes theinteraction between elasticity and strength (G′=stress/strain) and, assuch, provides a quantitative measurement of a composition's hardness orsoftness. The elastic modulus of an object is defined as the slope ofits stress-strain curve in the elastic deformation region:λ=stress/strain, where A is the elastic modulus in Pascal's; stress isthe force causing the deformation divided by the area to which the forceis applied; and strain is the ratio of the change caused by the stressto the original state of the object. Although depending on the speed atwhich the force is applied, a stiffer composition will have a higherelastic modulus and it will take a greater force to deform the materiala given distance, such as, e.g., an injection. Specifying how stressesare to be measured, including directions, allows for many types ofelastic moduli to be defined. The three primary elastic moduli aretensile modulus, shear modulus, and bulk modulus.

Viscous modulus is also known as the loss modulus because it describesthe energy that is lost as viscous dissipation. Tan δ is the ratio ofthe viscous modulus and the elastic modulus, tan δ=G″/G′. Falcone,supra, 2009. For tan δ values disclosed in the present specification, atan δ is obtained from the dynamic modulus at a frequency of 1 Hz. Alower tan δ corresponds to a stiffer, harder, or more elasticcomposition.

In another embodiment, a hydrogel composition disclosed herein exhibitsan elastic modulus. In aspects of this embodiment, a hydrogelcomposition exhibits an elastic modulus of, e.g., about 25 Pa, about 50Pa, about 75 Pa, about 100 Pa, about 125 Pa, about 150 Pa, about 175 Pa,about 200 Pa, about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa,about 450 Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa,about 700 Pa, about 750 Pa, about 800 Pa, about 850 Pa, about 900 Pa,about 950 Pa, about 1,000 Pa, about 1,200 Pa, about 1,300 Pa, about1,400 Pa, about 1,500 Pa, about 1,600 Pa, about 1700 Pa, about 1800 Pa,about 1900 Pa, about 2,000 Pa, about 2,100 Pa, about 2,200 Pa, about2,300 Pa, about 2,400 Pa, or about 2,500 Pa. In other aspects of thisembodiment, a hydrogel composition exhibits an elastic modulus of, e.g.,at least 25 Pa, at least 50 Pa, at least 75 Pa, at least 100 Pa, atleast 125 Pa, at least 150 Pa, at least 175 Pa, at least 200 Pa, atleast 250 Pa, at least 300 Pa, at least 350 Pa, at least 400 Pa, atleast 450 Pa, at least 500 Pa, at least 550 Pa, at least 600 Pa, atleast 650 Pa, at least 700 Pa, at least 750 Pa, at least 800 Pa, atleast 850 Pa, at least 900 Pa, at least 950 Pa, at least 1,000 Pa, atleast 1,200 Pa, at least 1,300 Pa, at least 1,400 Pa, at least 1,500 Pa,at least 1,600 Pa, at least 1700 Pa, at least 1800 Pa, at least 1900 Pa,at least 2,000 Pa, at least 2,100 Pa, at least 2,200 Pa, at least 2,300Pa, at least 2,400 Pa, or at least 2,500 Pa. In yet other aspects ofthis embodiment, a hydrogel composition exhibits an elastic modulus of,e.g., at most 25 Pa, at most 50 Pa, at most 75 Pa, at most 100 Pa, atmost 125 Pa, at most 150 Pa, at most 175 Pa, at most 200 Pa, at most 250Pa, at most 300 Pa, at most 350 Pa, at most 400 Pa, at most 450 Pa, atmost 500 Pa, at most 550 Pa, at most 600 Pa, at most 650 Pa, at most 700Pa, at most 750 Pa, at most 800 Pa, at most 850 Pa, at most 900 Pa, atmost 950 Pa, at most 1,000 Pa, at most 1,200 Pa, at most 1,300 Pa, atmost 1,400 Pa, at most 1,500 Pa, or at most 1,600 Pa. In still otheraspects of this embodiment, a hydrogel composition exhibits an elasticmodulus of, e.g., about 25 Pa to about 150 Pa, about 25 Pa to about 300Pa, about 25 Pa to about 500 Pa, about 25 Pa to about 800 Pa, about 125Pa to about 300 Pa, about 125 Pa to about 500 Pa, about 125 Pa to about800 Pa, about 500 Pa to about 1,600 Pa, about 600 Pa to about 1,600 Pa,about 700 Pa to about 1,600 Pa, about 800 Pa to about 1,600 Pa, about900 Pa to about 1,600 Pa, about 1,000 Pa to about 1,600 Pa, about 1,100Pa to about 1,600 Pa, about 1,200 Pa to about 1,600 Pa, about 500 Pa toabout 2,500 Pa, about 1,000 Pa to about 2,500 Pa, about 1,500 Pa toabout 2,500 Pa, about 2,000 Pa to about 2,500 Pa, about 1,300 Pa toabout 1,600 Pa, about 1,400 Pa to about 1,700 Pa, about 1,500 Pa toabout 1,800 Pa, about 1,600 Pa to about 1,900 Pa, about 1,700 Pa toabout 2,000 Pa, about 1,800 Pa to about 2,100 Pa, about 1,900 Pa toabout 2,200 Pa, about 2,000 Pa to about 2,300 Pa, about 2,100 Pa toabout 2,400 Pa, or about 2,200 Pa to about 2,500 Pa.

In another embodiment, a hydrogel composition disclosed herein exhibitsa viscous modulus. In aspects of this embodiment, a hydrogel compositionexhibits a viscous modulus of, e.g., about 10 Pa, about 20 Pa, about 30Pa, about 40 Pa, about 50 Pa, about 60 Pa, about 70 Pa, about 80 Pa,about 90 Pa, about 100 Pa, about 150 Pa, about 200 Pa, about 250 Pa,about 300 Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa,about 550 Pa, about 600 Pa, about 650 Pa, or about 700 Pa. In otheraspects of this embodiment, a hydrogel composition exhibits a viscousmodulus of, e.g., at most 10 Pa, at most 20 Pa, at most 30 Pa, at most40 Pa, at most 50 Pa, at most 60 Pa, at most 70 Pa, at most 80 Pa, atmost 90 Pa, at most 100 Pa, at most 150 Pa, at most 200 Pa, at most 250Pa, at most 300 Pa, at most 350 Pa, at most 400 Pa, at most 450 Pa, atmost 500 Pa, at most 550 Pa, at most 600 Pa, at most 650 Pa, or at most700 Pa. In yet other aspects of this embodiment, a hydrogel compositionexhibits a viscous modulus of, e.g., about 10 Pa to about 30 Pa, about10 Pa to about 50 Pa, about 10 Pa to about 100 Pa, about 10 Pa to about150 Pa, about 70 Pa to about 100 Pa, about 50 Pa to about 350 Pa, about150 Pa to about 450 Pa, about 250 Pa to about 550 Pa, about 350 Pa toabout 700 Pa, about 50 Pa to about 150 Pa, about 100 Pa to about 200 Pa,about 150 Pa to about 250 Pa, about 200 Pa to about 300 Pa, about 250 Pato about 350 Pa, about 300 Pa to about 400 Pa, about 350 Pa to about 450Pa, about 400 Pa to about 500 Pa, about 450 Pa to about 550 Pa, about500 Pa to about 600 Pa, about 550 Pa to about 650 Pa, or about 600 Pa toabout 700 Pa.

In another embodiment, a hydrogel composition disclosed herein exhibitsa tan δ. In aspects of this embodiment, a hydrogel composition exhibitsa tan δ of, e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5,about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8,about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, orabout 2.5. In other aspects of this embodiment, a hydrogel compositionexhibits a tan δ of, e.g., at most 0.1, at most 0.2, at most 0.3, atmost 0.4, at most 0.5, at most 0.6, at most 0.7, at most 0.8, at most0.9, at most 1.0, at most 1.1, at most 1.2, at most 1.3, at most 1.4, atmost 1.5, at most 1.6, at most 1.7, at most 1.8, at most 1.9, at most2.0, at most 2.1, at most 2.2, at most 2.3, at most 2.4, or at most 2.5.In yet other aspects of this embodiment, a hydrogel composition exhibitsa tan δ of, e.g., about 0.1 to about 0.3, about 0.3 to about 0.5, about0.5 to about 0.8, about 1.1 to about 1.4, about 1.4 to about 1.7, about0.3 to about 0.6, about 0.1 to about 0.5, about 0.5 to about 0.9, about0.1 to about 0.6, about 0.1 to about 1.0, about 0.5 to about 1.5, about1.0 to about 2.0, or about 1.5 to about 2.5.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein having a transparency and/or translucency.Optical transparency is the physical property of allowing visible lightto pass through a material, whereas translucency (also calledtranslucence or translucidity) only allows light to pass throughdiffusely. The opposite property is opacity. Transparent materials areclear, while translucent ones cannot be seen through clearly. Thehydrogels disclosed herein are preferably optically transparent or atleast translucent.

In an embodiment, a hydrogel composition disclosed herein is opticallytranslucent. In aspects of this embodiment, a hydrogel compositiondiffusely transmits, e.g., about 75% of the light, about 80% of thelight, about 85% of the light, about 90% of the light, about 95% of thelight, or about 100% of the light. In other aspects of this embodiment,a hydrogel composition diffusely transmits, e.g., at least 75% of thelight, at least 80% of the light, at least 85% of the light, at least90% of the light, or at least 95% of the light. In yet other aspects ofthis embodiment, a hydrogel composition diffusely transmits, e.g., about75% to about 100% of the light, about 80% to about 100% of the light,about 85% to about 100% of the light, about 90% to about 100% of thelight, or about 95% to about 100% of the light. In an embodiment, ahydrogel composition disclosed herein is optically transparent andtransmits 100% of visible light.

A hydrogel composition disclosed herein may be further processed bypulverizing the hydrogel into particles and optionally mixed with acarrier phase such as, e.g., water or a saline solution to form aninjectable or topical substance like a solution, oil, lotion, gel,ointment, cream, slurry, salve, or paste. As such, the disclosedhydrogel compositions may be monophasic or multiphasic compositions. Ahydrogel may be milled to a particle size from about 10 μm to about 1000μm in diameter, such as about 15 μm to about 30 μm, about 50 μm to about75 μm, about 100 μm to about 150 μm, about 200 μm to about 300 μm, about450 μm to about 550 μm, about 600 μm to about 700 μm, about 750 μm toabout 850 μm, or about 900 μm to about 1,000 μm.

Aspects of the present specification provide, in part, a compositiondisclosed herein is injectable. As used herein, the term “injectable”refers to a material having the properties necessary to administer thecomposition into a skin region of an individual using an injectiondevice with a fine needle. As used herein, the term “fine needle” refersto a needle that is 27 gauge or smaller. Injectability of a compositiondisclosed herein can be accomplished by sizing the hydrogel particles asdiscussed above.

In aspect of this embodiment, a hydrogel composition disclosed herein isinjectable through a fine needle. In other aspects of this embodiment, ahydrogel composition disclosed herein is injectable through a needle of,e.g., about 27 gauge, about 30 gauge, or about 32 gauge. In yet otheraspects of this embodiment, a hydrogel composition disclosed herein isinjectable through a needle of, e.g., 22 gauge or smaller, 27 gauge orsmaller, 30 gauge or smaller, or 32 gauge or smaller. In still otheraspects of this embodiment, a hydrogel composition disclosed herein isinjectable through a needle of, e.g., about 22 gauge to about 35 gauge,22 gauge to about 34 gauge, 22 gauge to about 33 gauge, 22 gauge toabout 32 gauge, about 22 gauge to about 27 gauge, or about 27 gauge toabout 32 gauge.

In aspects of this embodiment, a hydrogel composition disclosed hereincan be injected with an extrusion force of about 60 N, about 55 N, about50 N, about 45 N, about 40 N, about 35 N, about 30 N, about 25 N, about20 N, or about 15 N at speeds of 100 mm/min. In other aspects of thisembodiment, a hydrogel composition disclosed herein can be injectedthrough a 27 gauge needle with an extrusion force of about 60 N or less,about 55 N or less, about 50 N or less, about 45 N or less, about 40 Nor less, about 35 N or less, about 30 N or less, about 25 N or less,about 20 N or less, about 15 N or less, about 10 N or less, or about 5 Nor less. In yet other aspects of this embodiment, a hydrogel compositiondisclosed herein can be injected through a 30 gauge needle with anextrusion force of about 60 N or less, about 55 N or less, about 50 N orless, about 45 N or less, about 40 N or less, about 35 N or less, about30 N or less, about 25 N or less, about 20 N or less, about 15 N orless, about 10 N or less, or about 5 N or less. In still other aspectsof this embodiment, a hydrogel composition disclosed herein can beinjected through a 32 gauge needle with an extrusion force of about 60 Nor less, about 55 N or less, about 50 N or less, about 45 N or less,about 40 N or less, about 35 N or less, about 30 N or less, about 25 Nor less, about 20 N or less, about 15 N or less, about 10 N or less, orabout 5 N or less.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits cohesivity. Cohesivity, alsoreferred to as cohesion cohesive attraction, cohesive force, orcompression force is a physical property of a material, caused by theintermolecular attraction between like-molecules within the materialthat acts to unite the molecules. Cohesivity is expressed in terms ofgrams-force (gmf). Cohesiveness is affected by, among other factors, themolecular weight ratio of the initial free glycosaminoglycan polymer,the degree of crosslinking of glycosaminoglycan polymers, the amount ofresidual free glycosaminoglycan polymers following crosslinking, and thepH of the hydrogel composition. A composition should be sufficientlycohesive as to remain localized to a site of administration.Additionally, in certain applications, a sufficient cohesiveness isimportant for a composition to retain its shape, and thus functionality,in the event of mechanical load cycling. As such, in one embodiment, ahydrogel composition disclosed herein exhibits cohesivity, on par withwater. In yet another embodiment, a hydrogel composition disclosedherein exhibits sufficient cohesivity to remain localized to a site ofadministration. In still another embodiment, a hydrogel compositiondisclosed herein exhibits sufficient cohesivity to retain its shape. Ina further embodiment, a hydrogel composition disclosed herein exhibitssufficient cohesivity to retain its shape and functionality.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits a physiologically-acceptableosmolarity. As used herein, the term “osmolarity” refers to theconcentration of osmotically active solutes in solution. As used herein,the term “a physiologically-acceptable osmolarity” refers to anosmolarity in accord with, or characteristic of, the normal functioningof a living organism. As such, administration of a hydrogel compositionas disclosed herein exhibits an osmolarity that has substantially nolong term or permanent detrimental effect when administered to a mammal.Osmolarity is expressed in terms of osmoles of osmotically active soluteper liter of solvent (Osmol/L or Osm/L). Osmolarity is distinct frommolarity because it measures moles of osmotically active soluteparticles rather than moles of solute. The distinction arises becausesome compounds can dissociate in solution, whereas others cannot. Theosmolarity of a solution can be calculated from the followingexpression: Osmol/L=Σφ_(i) η_(i) C_(i), where φ is the osmoticcoefficient, which accounts for the degree of non-ideality of thesolution; n is the number of particles (e.g. ions) into which a moleculedissociates; and C is the molar concentration of the solute; and i isthe index representing the identity of a particular solute. Theosmolarity of a hydrogel composition disclosed herein can be measuredusing a conventional method that measures solutions.

In an embodiment, a hydrogel composition disclosed herein exhibits aphysiologically-acceptable osmolarity. As used herein, the term“osmolality” refers to the concentration of osmotically active solutesper kilo of solvent in the body. As used herein, the term “aphysiologically-acceptable osmolality” refers to an osmolality in accordwith, or characteristic of, the normal functioning of a living organism.As such, administration of a hydrogel composition disclosed hereinexhibits an osmolality that has substantially no long term or permanentdetrimental effect when administered to a mammal. Osmolality isexpressed in terms of osmoles of osmotically active solute per kilogramof solvent (osmol/kg or Osm/kg) and is equal to the sum of themolalities of all the solutes present in that solution. The osmolalityof a solution can be measured using an osmometer. The most commonly usedinstrument in modern laboratories is a freezing point depressionosmometer. This instruments measure the change in freezing point thatoccurs in a solution with increasing osmolality (freezing pointdepression osmometer) or the change in vapor pressure that occurs in asolution with increasing osmolality (vapor pressure depressionosmometer).

In aspects of this embodiment, a hydrogel composition exhibits anosmolarity of, e.g., about 100 mOsm/L, about 150 mOsm/L, about 200mOsm/L, about 250 mOsm/L, about 300 mOsm/L, about 350 mOsm/L, about 400mOsm/L, about 450 mOsm/L, or about 500 mOsm/L. In other aspects of thisembodiment, a hydrogel composition exhibits an osmolarity of, e.g., atleast 100 mOsm/L, at least 150 mOsm/L, at least 200 mOsm/L, at least 250mOsm/L, at least 300 mOsm/L, at least 350 mOsm/L, at least 400 mOsm/L,at least 450 mOsm/L, or at least 500 mOsm/L. In yet other aspects ofthis embodiment, a hydrogel composition exhibits an osmolarity of, e.g.,at most 100 mOsm/L, at most 150 mOsm/L, at most 200 mOsm/L, at most 250mOsm/L, at most 300 mOsm/L, at most 350 mOsm/L, at most 400 mOsm/L, atmost 450 mOsm/L, or at most 500 mOsm/L. In still other aspects of thisembodiment, a hydrogel composition exhibits an osmolarity of, e.g.,about 100 mOsm/L to about 500 mOsm/L, about 200 mOsm/L to about 500mOsm/L, about 200 mOsm/L to about 400 mOsm/L, about 300 mOsm/L to about400 mOsm/L, about 270 mOsm/L to about 390 mOsm/L, about 225 mOsm/L toabout 350 mOsm/L, about 250 mOsm/L to about 325 mOsm/L, about 275 mOsm/Lto about 300 mOsm/L, or about 285 mOsm/L to about 290 mOsm/L.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits substantial stability. Asused herein, the term “stability” or “stable” when referring to ahydrogel composition disclosed herein refers to a composition that isnot prone to degrading, decomposing, or breaking down to any substantialor significant degree while stored before administration to anindividual. As used herein, the term “substantial heat stability”,“substantially heat stable”, “autoclave stable”, or “steam sterilizationstable” refers to a hydrogel composition disclosed herein that issubstantially stable when subjected to a heat treatment as disclosedherein.

Stability of a hydrogel composition disclosed herein can be determinedby subjecting a hydrogel composition to a heat treatment, such as, e.g.,steam sterilization at normal pressure or under pressure (e.g.,autoclaving). Preferably the heat treatment is carried out at atemperature of at least about 100° C. for between about one minute andabout 10 minutes. Substantial stability of a hydrogel compositiondisclosed herein can be evaluated 1) by determining the change in theextrusion force (ΔF) of a hydrogel composition disclosed herein aftersterilization, where the change in extrusion force less 2N is indicativeof a substantially stable hydrogel composition as measured by (theextrusion force of a hydrogel composition with the specified additives)minus (the extrusion force of the a hydrogel composition without theadded additives); and/or 2) by determining the change in rheologicalproperties of a hydrogel composition disclosed herein aftersterilization, where the change in tan δ 1 Hz of less than 0.1 isindicative of a substantially stable hydrogel composition as measured by(tan δ 1 Hz of gel formulation with additives) minus (tan δ 1 Hz of gelformulation without additives). As such, a substantially stable hydrogelcomposition disclosed herein retains one or more of the followingcharacteristics after sterilization: homogeneousness, extrusion force,cohesiveness, hyaluronan concentration, agent(s) concentration,osmolarity, pH, or other rheological characteristics desired by thehydrogel before the heat treatment.

In an embodiment, a hydrogel composition comprising a glycosaminoglycanpolymer and the at least one agent disclosed herein is processed using aheat treatment that maintains the desired hydrogel properties disclosedherein. In aspects of this embodiment, a hydrogel composition comprisinga glycosaminoglycan polymer and the at least one agent disclosed hereinis processed using a heat treatment of, e.g., about 100° C., about 105°C., about 110° C., about 115° C., about 120° C., about 125° C., or about130° C. In other aspects of this embodiment, a hydrogel compositioncomprising a glycosaminoglycan polymer and the at least one agentdisclosed herein is processed using a heat treatment of, e.g., at least100° C., at least 105° C., at least 110° C., at least 115° C., at least120° C., at least 125° C., or at least 130° C. In yet other aspects ofthis embodiment, a hydrogel composition comprising a glycosaminoglycanpolymer and the at least one agent disclosed herein is processed using aheat treatment of, e.g., about 100° C. to about 120° C., about 100° C.to about 125° C., about 100° C. to about 130° C., about 100° C. to about135° C., about 110° C. to about 120° C., about 110° C. to about 125° C.,about 110° C. to about 130° C., about 110° C. to about 135° C., about120° C. to about 125° C., about 120° C. to about 130° C., about 120° C.to about 135° C., about 125° C. to about 130° C., or about 125° C. toabout 135° C.

Long term stability of a hydrogel composition disclosed herein can bedetermined by subjecting a hydrogel composition to a heat treatment,such as, e.g., storage in an about 45° C. environment for about 60 days.Long term stability of a hydrogel composition disclosed herein can beevaluated 1) by assessing the clarity and color of a hydrogelcomposition after the 45° C. heat treatment, with a clear and uncoloredhydrogel composition being indicative of a substantially stable hydrogelcomposition; 2) by determining the change in the extrusion force (ΔF) ofa hydrogel composition disclosed herein after the 45° C. heat treatment,where the change in extrusion force less 2N is indicative of asubstantially stable hydrogel composition as measured by (the extrusionforce of a hydrogel composition with the specified additives before the45° C. heat treatment) minus (the extrusion force of the a hydrogelcomposition with the specified additives after the 45° C. heattreatment); and/or 3) by determining the change in rheologicalproperties of a hydrogel composition disclosed herein aftersterilization, where the change in tan δ 1 Hz of less than 0.1 isindicative of a substantially stable hydrogel composition as measured by(tan δ 1 Hz of gel formulation with the specified additives before the45° C. heat treatment) minus (tan δ 1 Hz of gel formulation with thespecified additives after the 45° C. heat treatment). As such, a longterm stability of a hydrogel composition disclosed herein is evaluatedby retention of one or more of the following characteristics after the45° C. heat treatment: clarity (transparency and translucency),homogeneousness, and cohesiveness.

In aspects of this embodiment, a hydrogel composition is substantiallystable at room temperature for, e.g., about 3 months, about 6 months,about 9 months, about 12 months, about 15 months, about 18 months, about21 months, about 24 months, about 27 months, about 30 months, about 33months, or about 36 months. In other aspects of this embodiment, ahydrogel composition is substantially stable at room temperature for,e.g., at least 3 months, at least 6 months, at least 9 months, at least12 months, at least 15 months, at least 18 months, at least 21 months,at least 24 months, at least 27 months, at least 30 months, at least 33months, or at least 36 months. In other aspects of this embodiment, ahydrogel composition is substantially stable at room temperature for,e.g., about 3 months to about 12 months, about 3 months to about 18months, about 3 months to about 24 months, about 3 months to about 30months, about 3 months to about 36 months, about 6 months to about 12months, about 6 months to about 18 months, about 6 months to about 24months, about 6 months to about 30 months, about 6 months to about 36months, about 9 months to about 12 months, about 9 months to about 18months, about 9 months to about 24 months, about 9 months to about 30months, about 9 months to about 36 months, about 12 months to about 18months, about 12 months to about 24 months, about 12 months to about 30months, about 12 months to about 36 months, about 18 months to about 24months, about 18 months to about 30 months, or about 18 months to about36 months.

The present compositions may optionally include, without limitation,other pharmaceutically acceptable components, including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, osmolality adjusting agents, emulsifying agents, wettingagents, and the like.

A pharmaceutically acceptable buffer is a buffer that can be used toprepare a hydrogel composition disclosed herein, provided that theresulting preparation is pharmaceutically acceptable. Non-limitingexamples of pharmaceutically acceptable buffers include acetate buffers,borate buffers, citrate buffers, neutral buffered salines, phosphatebuffers, and phosphate buffered salines. Any concentration of apharmaceutically acceptable buffer can be useful in formulating apharmaceutical composition disclosed herein, with the proviso that atherapeutically effective amount of the active ingredient is recoveredusing this effective concentration of buffer. Non-limiting examples ofconcentrations of physiologically-acceptable buffers occur within therange of about 0.1 mM to about 900 mM. The pH of pharmaceuticallyacceptable buffers may be adjusted, provided that the resultingpreparation is pharmaceutically acceptable. It is understood that acidsor bases can be used to adjust the pH of a pharmaceutical composition asneeded. Any buffered pH level can be useful in formulating apharmaceutical composition, with the proviso that a therapeuticallyeffective amount of the matrix polymer active ingredient is recoveredusing this effective pH level. Non-limiting examples ofphysiologically-acceptable pH occur within the range of about pH 5.0 toabout pH 8.5. For example, the pH of a hydrogel composition disclosedherein can be about 5.0 to about 8.0, or about 6.5 to about 7.5, about7.0 to about 7.4, or about 7.1 to about 7.3.

Pharmaceutically acceptable preservatives include, without limitation,sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Pharmaceutically acceptablepreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate, a stabilized oxy chloro composition, such as, e.g., PURITE®(Allergan, Inc. Irvine, Calif.) and chelants, such as, e.g., DTPA orDTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide.

Pharmaceutically acceptable tonicity adjustors useful in a hydrogelcomposition disclosed herein include, without limitation, salts such as,e.g., sodium chloride and potassium chloride; and glycerin. Thecomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition disclosedherein. Other non-limiting examples of pharmacologically acceptablecomponents can be found in, e.g., Ansel, supra, (1999); Gennaro, supra,(2000); Hardman, supra, (2001); and Rowe, supra, (2003), each of whichis hereby incorporated by reference in its entirety.

Aspects of the present specification provide, in part, a method oftreating a soft tissue condition of an individual by administering ahydrogel composition disclosed herein. As used herein, the term“treating,” refers to reducing or eliminating in an individual acosmetic or clinical symptom of a soft tissue condition characterized bya soft tissue imperfection, defect, disease, and/or disorder; ordelaying or preventing in an individual the onset of a cosmetic orclinical symptom of a condition characterized by a soft tissueimperfection, defect, disease, and/or disorder. For example, the term“treating” can mean reducing a symptom of a condition characterized by asoft tissue defect, disease, and/or disorder by, e.g., at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100%. The effectiveness of ahydrogel composition disclosed herein in treating a conditioncharacterized by a soft tissue defect, disease, and/or disorder can bedetermined by observing one or more cosmetic, clinical symptoms, and/orphysiological indicators associated with the condition. An improvementin a soft tissue defect, disease, and/or disorder also can be indicatedby a reduced need for a concurrent therapy. Those of skill in the artwill know the appropriate symptoms or indicators associated withspecific soft tissue defect, disease, and/or disorder and will know howto determine if an individual is a candidate for treatment with acompound or composition disclosed herein.

A hydrogel composition in accordance with the invention is administeredto an individual. An individual is typically a human being of any age,gender or race. Typically, any individual who is a candidate for aconventional procedure to treat a soft tissue condition is a candidatefor a method disclosed herein. Although a subject experiencing the signsof aging skin is an adult, subjects experiencing premature aging orother skin conditions suitable for treatment (for example, a scar) canalso be treated with a hydrogel composition disclosed herein. Inaddition, the presently disclosed hydrogel compositions and methods mayapply to individuals seeking a small/moderate enlargement, shape changeor contour alteration of a body part or region, which may not betechnically possible or aesthetically acceptable with existing softtissue implant technology. Pre-operative evaluation typically includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure.

The hydrogel composition and methods disclosed herein are useful intreating a soft tissue condition. A soft tissue condition includes,without limitation, a soft tissue imperfection, defect, disease, and/ordisorder. Non-limiting examples of a soft tissue condition includebreast imperfection, defect, disease and/or disorder, such as, e.g., abreast augmentation, a breast reconstruction, mastopexy, micromastia,thoracic hypoplasia, Poland's syndrome, defects due to implantcomplications like capsular contraction and/or rupture; a facialimperfection, defect, disease or disorder, such as, e.g., a facialaugmentation, a facial reconstruction, a mesotherapy, Parry-Rombergsyndrome, lupus erythematosus profundus, dermal divots, scars, sunkenchecks, thin lips, nasal imperfections or defects, retro-orbitalimperfections or defects, a facial fold, line and/or wrinkle like aglabellar line, a nasolabial line, a perioral line, and/or a marionetteline, and/or other contour deformities or imperfections of the face; aneck imperfection, defect, disease or disorder; a skin imperfection,defect, disease and/or disorder; other soft tissue imperfections,defects, diseases and/or disorders, such as, e.g., an augmentation or areconstruction of the upper arm, lower arm, hand, shoulder, back, torsoincluding abdomen, buttocks, upper leg, lower leg including calves, footincluding plantar fat pad, eye, genitals, or other body part, region orarea, or a disease or disorder affecting these body parts, regions orareas; urinary incontinence, fecal incontinence, other forms ofincontinence; and gastroesophageal reflux disease (GERD). As usedherein, the term “mesotherapy” refers to a non-surgical cosmetictreatment technique of the skin involving intra-epidermal, intra-dermal,and/or subcutaneous injection of an agent administered as small multipledroplets into the epidermis, dermo-epidermal junction, and/or thedermis.

The amount of a hydrogel composition used with any of the methods asdisclosed herein will typically be determined based on the alterationand/or improvement desired, the reduction and/or elimination of a softtissue condition symptom desired, the clinical and/or cosmetic effectdesired by the individual and/or physician, and the body part or regionbeing treated. The effectiveness of composition administration may bemanifested by one or more of the following clinical and/or cosmeticmeasures: altered and/or improved soft tissue shape, altered and/orimproved soft tissue size, altered and/or improved soft tissue contour,altered and/or improved tissue function, tissue ingrowth support and/ornew collagen deposition, sustained engraftment of composition, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign material.

Effectiveness of the compositions and methods in treating a facial softtissue may be manifested by one or more of the following clinical and/orcosmetic measures: increased size, shape, and/or contour of facialfeature like increased size, shape, and/or contour of lip, cheek or eyeregion; altered size, shape, and/or contour of facial feature likealtered size, shape, and/or contour of lip, cheek or eye region shape;reduction or elimination of a wrinkle, fold or line in the skin;resistance to a wrinkle, fold or line in the skin; rehydration of theskin; increased elasticity to the skin; reduction or elimination of skinroughness; increased and/or improved skin tautness; reduction orelimination of stretch lines or marks; increased and/or improved skintone, shine, brightness and/or radiance; increased and/or improved skincolor, reduction or elimination of skin paleness; sustained engraftmentof composition; decreased side effects; improved patient satisfactionand/or quality of life.

As yet another example, for urinary incontinence procedures,effectiveness of the compositions and methods for sphincter support maybe manifested by one or more of the following clinical measures:decreased frequency of incontinence, sustained engraftment, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign filler.

In aspects of this embodiment, the amount of a hydrogel compositionadministered is, e.g., about 0.01 g, about 0.05 g, about 0.1 g, about0.5 g, about 1 g, about 5 g, about 10 g, about 20 g, about 30 g, about40 g, about 50 g, about 60 g, about 70 g, about 80 g, about 90 g, about100 g, about 150 g, or about 200 g. In other aspects of this embodiment,the amount of a hydrogel composition administered is, e.g., about 0.01 gto about 0.1 g, about 0.1 g to about 1 g, about 1 g to about 10 g, about10 g to about 100 g, or about 50 g to about 200 g. In yet other aspectsof this embodiment, the amount of a hydrogel composition administeredis, e.g., about 0.01 mL, about 0.05 mL, about 0.1 mL, about 0.5 mL,about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40mL, about 50 mL, about 60 mL, about 70 g, about 80 mL, about 90 mL,about 100 mL, about 150 mL, or about 200 mL. In other aspects of thisembodiment, the amount of a hydrogel composition administered is, e.g.,about 0.01 mL to about 0.1 mL, about 0.1 mL to about 1 mL, about 1 mL toabout 10 mL, about 10 mL to about 100 mL, or about 50 mL to about 200mL.

The duration of treatment will typically be determined based on thecosmetic and/or clinical effect desired by the individual and/orphysician and the body part or region being treated. In aspects of thisembodiment, administration of a hydrogel composition disclosed hereincan treat a soft tissue condition for, e.g., about 6 months, about 7months, about 8 months, about 9 months, about 10 months, about 11months, about 12 months, about 13 months, about 14 months, about 15months, about 18 months, or about 24 months. In other aspects of thisembodiment, administration of a hydrogel composition disclosed hereincan treat a soft tissue condition for, e.g., at least 6 months, at least7 months, at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months, at least 13 months, at least 14months, at least 15 months, at least 18 months, or at least 24 months.In yet aspects of this embodiment, administration of a hydrogelcomposition disclosed herein can treat a soft tissue condition for,e.g., about 6 months to about 12 months, about 6 months to about 15months, about 6 months to about 18 months, about 6 months to about 21months, about 6 months to about 24 months, about 9 months to about 12months, about 9 months to about 15 months, about 9 months to about 18months, about 9 months to about 21 months, about 6 months to about 24months, about 12 months to about 15 months, about 12 months to about 18months, about 12 months to about 21 months, about 12 months to about 24months, about 15 months to about 18 months, about 15 months to about 21months, about 15 months to about 24 months, about 18 months to about 21months, about 18 months to about 24 months, or about 21 months to about24 months.

Aspects of the present specification provide, in part, administering ahydrogel composition disclosed herein. As used herein, the term“administering” means any delivery mechanism that provides a compositiondisclosed herein to an individual that potentially results in aclinically, therapeutically, or experimentally beneficial result. Theactual delivery mechanism used to administer a composition to anindividual can be determined by a person of ordinary skill in the art bytaking into account factors, including, without limitation, the type ofskin condition, the location of the skin condition, the cause of theskin condition, the severity of the skin condition, the degree of reliefdesired, the duration of relief desired, the particular compositionused, the rate of excretion of the particular composition used, thepharmacodynamics of the particular composition used, the nature of theother compounds included in the particular composition used, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, or any combination thereof. In an aspect ofthis embodiment, a composition disclosed herein is administered to askin region of an individual by injection.

The route of administration of a hydrogel composition to an individualpatient will typically be determined based on the cosmetic and/orclinical effect desired by the individual and/or physician and the bodypart or region being treated. A composition disclosed herein may beadministered by any means known to persons of ordinary skill in the artincluding, without limitation, syringe with needle, a pistol (forexample, a hydropneumatic-compression pistol), catheter, topically, orby direct surgical implantation. The hydrogel composition disclosedherein can be administered into a skin region such as, e.g., a dermalregion or a hypodermal region. For example, a hydrogel compositiondisclosed herein can be injected utilizing needles with a diameter ofabout 0.26 mm to about 0.4 mm and a length ranging from about 4 mm toabout 14 mm. Alternately, the needles can be 21 to 32 G and have alength of about 4 mm to about 70 mm. Preferably, the needle is asingle-use needle. The needle can be combined with a syringe, catheter,and/or a pistol.

In addition, a composition disclosed herein can be administered once, orover a plurality of times. Ultimately, the timing used will followquality care standards. For example, a hydrogel composition disclosedherein can be administered once or over several sessions with thesessions spaced apart by a few days, or weeks. For instance, anindividual can be administered a hydrogel composition disclosed hereinevery 1, 2, 3, 4, 5, 6, or 7 days or every 1, 2, 3, or 4 weeks. Theadministration a hydrogel composition disclosed herein to an individualcan be on a monthly or bi-monthly basis or administered every 3, 6, 9,or 12 months.

Aspects of the present specification provide, in part, a dermal region.As used herein, the term “dermal region” refers to the region of skincomprising the epidermal-dermal junction and the dermis including thesuperficial dermis (papillary region) and the deep dermis (reticularregion). The skin is composed of three primary layers: the epidermis,which provides waterproofing and serves as a barrier to infection; thedermis, which serves as a location for the appendages of skin; and thehypodermis (subcutaneous adipose layer). The epidermis contains no bloodvessels, and is nourished by diffusion from the dermis. The main type ofcells which make up the epidermis are keratinocytes, melanocytes,Langerhans cells and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many Mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the Stratum basale of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Tattoo ink is held in thedermis. Stretch marks from pregnancy are also located in the dermis.

The hypodermis lies below the dermis. Its purpose is to attach thedermal region of the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

In an aspect of this embodiment, a hydrogel composition disclosed hereinis administered to a skin region of an individual by injection into adermal region or a hypodermal region. In aspects of this embodiment, ahydrogel composition disclosed herein is administered to a dermal regionof an individual by injection into, e.g., an epidermal-dermal junctionregion, a papillary region, a reticular region, or any combinationthereof.

Advantageously, some of the present compositions are especially usefuland effective in reducing appearance of fine lines, for example, in thinskin regions, of a patient. For example, methods are provided for fineline treatment comprising the steps of administering to the patient adermal filler composition as described elsewhere herein, at a depth ofno greater than about 1 mm.

Other aspects of the present specification disclose, in part, a methodof treating a skin condition comprises the step of administering to anindividual suffering from a skin condition a hydrogel compositiondisclosed herein, wherein the administration of the composition improvesthe skin condition, thereby treating the skin condition. In an aspect ofthis embodiment, a skin condition is a method of treating skindehydration comprises the step of administering to an individualsuffering from skin dehydration a hydrogel composition disclosed herein,wherein the administration of the composition rehydrates the skin,thereby treating skin dehydration. In another aspect of this embodiment,a method of treating a lack of skin elasticity comprises the step ofadministering to an individual suffering from a lack of skin elasticitya hydrogel composition disclosed herein, wherein the administration ofthe composition increases the elasticity of the skin, thereby treating alack of skin elasticity. In yet another aspect of this embodiment, amethod of treating skin roughness comprises the step of administering toan individual suffering from skin roughness a hydrogel compositiondisclosed herein, wherein the administration of the compositiondecreases skin roughness, thereby treating skin roughness. In stillanother aspect of this embodiment, a method of treating a lack of skintautness comprises the step of administering to an individual sufferingfrom a lack of skin tautness a hydrogel composition disclosed herein,wherein the administration of the composition makes the skin tauter,thereby treating a lack of skin tautness.

In a further aspect of this embodiment, a method of treating a skinstretch line or mark comprises the step of administering to anindividual suffering from a skin stretch line or mark a hydrogelcomposition disclosed herein, wherein the administration of thecomposition reduces or eliminates the skin stretch line or mark, therebytreating a skin stretch line or mark. In another aspect of thisembodiment, a method of treating skin paleness comprises the step ofadministering to an individual suffering from skin paleness a hydrogelcomposition disclosed herein, wherein the administration of thecomposition increases skin tone or radiance, thereby treating skinpaleness. In another aspect of this embodiment, a method of treatingskin wrinkles comprises the step of administering to an individualsuffering from skin wrinkles a hydrogel composition disclosed herein,wherein the administration of the composition reduces or eliminates skinwrinkles, thereby treating skin wrinkles. In yet another aspect of thisembodiment, a method of treating skin wrinkles comprises the step ofadministering to an individual a hydrogel composition disclosed herein,wherein the administration of the composition makes the skin resistantto skin wrinkles, thereby treating skin wrinkles.

In some embodiments, the dermal fillers have a sustainedbioavailability. For example, dermal fillers are provided which, whenintroduced into the skin of a human being, (for example, intradermallyor subdermally into a human being for the correction of soft tissuedefects of voids in the face), release ascorbic acid (or other vitamin)into the human being for at least about 1 months and up to about 20months or more.

For example, to predict a sustained Vitamin C efficacy in coordinatewith filler duration, an estimation on conjugated degree is made. Thisestimation was based on the formulation of AA2G conjugation to HA viaetherification. The formulation is stable under physiological conditionsbut start to release of Ascorbic acid (AsA) by α-glucosidase which isattached to the cell membrane. Release of AsA happens at the filler/cellinterface due to the fact that α-glucosidase is attached to cellmembrane. Further release of AsA from HA-AA2G will be accompanied by HAdegradation to make AA2G available to fibroblasts. The release of AsA isthus depending on AA2G conjugation degree and duration of HA. A gel withconjugation degree of 5 mol % approximately could release active VitaminC in a period of at least up to 1 month, for example, between 3˜5months; a gel with 10 mol % conjugation degree could release activeVitamin C in a period up to 6˜8 months; a gel with 15 mol % conjugationdegree could release active Vitamin C in a period up to 10˜ months; 30mol % up to one and half years.

Conjugation Total AsA Calculated number degree (mol %) available*(mM)(months)** 3 2.13 2.8 5 3.55 3.1 1 7.10 6.3 15 10.65 9.4 25 17.75 15.730 21.13 18.8 *Based on parameters of Gels: volume, 0.1 cm³;concentration, 24 mg/ml. (0.1 × 24 × 3% × 1000)/(338*0.1) = 2.13 (mM)**Assumptions: AsA is released at a constant rate. Effectiveconcentration of AsA is 0.05 mM and maintains effective >2 days 2.13*2/(0.05*30) = 2.8 (months)

In an embodiment of the invention, a dermal filler is providedcomprising hyaluronic acid crosslinked with a Star-PEG epoxide andhaving a vitamin C derivative (for example, one of AA2G (Ascorbic acid2-Glucoside), Vitagen (3-aminopropyl-L-ascorbyl phosphate) and SAP(sodium ascorbyl phosphate) conjugated to the hyaluronic acid with adegree of conjugation of between about 3 mol % and about 40 mol %.

Methods of making this dermal filler include reacting pentaerythritolglycidal ether (Star-PEG epoxide) with ascorbic acid 2-Glucoside (AA2G)at a ratio, reaction temperature and reaction time suitable forachieving a composition containing AA2G capped by 4-arm epoxides (AA2G-4arm epoxides), unreacted 4-arm epoxides and free AA2G. The 4 arm epoxidecapped AA2G (AA2G-4 arm epoxides) is conjugated to hyaluronic acid viathe epoxyl group. The unreacted 4 arm epoxides serves as a crosslinkerto crosslink hyaluronic acid and as a conjugation agent to furtherconjugate AA2G.

In another embodiment of the invention, a dermal filler is providedcomprising hyaluronic acid crosslinked with BDDE and having a vitamin Cderivative (for example, one of AA2G (Ascorbic acid 2-Glucoside),Vitagen (3-am inopropyl-L-ascorbyl phosphate) and SAP (sodium ascorbylphosphate) conjugated to the hyaluronic acid with a degree ofconjugation of between about 3 mol % and about 10 mol %.

Methods of making this dermal filler include reacting BDDE with ascorbicacid 2-Glucoside (AA2G) at a ratio, reaction temperature and reactiontime suitable for achieve a composition containing AA2G capped by BDDE(AA2G-BDDE), unreacted BDDE and free AA2G. The BDDE capped AA2G(AA2G-BDDE) is conjugated to hyaluronic acid via the epoxyl group. Theunreacted BDDE serves as a crosslinker to crosslink hyaluronic acid andas a conjugation agent to further conjugate AA2G.

FIG. 9 is a Table showing the effect of a-glucosidase concentration onAsA release from AA2G-PBS solution. The conversion of AA2G to AsAdepends on the concentration of α-glycosidase. AA2G converted AsA almostcompletely in 15 minutes when α-glycosidase concentration is 6.3 unitper gram gel. When α-glycosidase concentration is 4.7 units per gramgel, It took 30 minutes to completely convert AA2G to AsA. Furtherdecease α-glycosidase concentration resulted in slow conversion of AA2Gto AsA.

FIG. 10 shows a representation of a release profile of free AsA fromconjugated dermal fillers in accordance with the invention (sustainedrelease) (AA2G conversion in mol % versus reaction time). AA2Gcompletely converted to AsA in AA2G/HA mix in 40 minutes. AA2H/HAconjugates showed a time dependence of AA2G conversion to AsA.

FIGS. 11A and 11B show additional release data for various dermalfillers in accordance with the invention. More specifically, conversionof AA2G to AsA in HA-AA2G gels is dependent on α-glycosidaseconcentration. High α-glycosidase concentration resulted in a fastconversion of AA2G to AsA. For a given α-glycosidase concentration,different formulations showed different profiles of AA2G to AsA.

In one aspect of the invention, dermal fillers are provided which areespecially effective in treating and eliminating the appearance of finelines, for example, relatively superficial, creases in the skin, forexample, but not limited to, fine lines near the eyes, the tear troughregion, forehead, periorbital, glabellar lines, etc.

The appearance of a blue discoloration at the skin site where a dermalfiller had been injected, (Tyndall effect) is a significant adverseevent experienced by some dermal filler patients. Tyndall effect is morecommon in patients treated for superficial fine line wrinkles.Embodiments of the present invention have been developed which providelong lasting, translucent fillers which can be injected superficially totreat fine lines and wrinkles, even in regions of relatively thin skin,without any resulting blue discoloration from Tyndall effect. Fine linesor superficial wrinkles are generally understood to be those wrinkles orcreases in skin that are typically found in regions of theface(forehead, lateral canthus, vermillion border/perioral lines) wherethe skin is thinnest, that is, the skin has a dermis thickness of lessthan 1 mm. On the forehead the average dermal thickness is about 0.95 mmfor normal skin and about 0.81 mm for wrinkled skin. Dermis around thelateral canthus is even thinner (e.g. about 0.61 mm for normal skin andabout 0.41 mm for wrinkled skin). The average outer diameter of a 30 or32 gauge needle (needles that are typically used for fine line gelapplication) is about 0.30 and about 0.24 mm.

The present invention provides a dermal filler composition such asdescribed elsewhere herein, which does not result in Tyndall effect. Forexample, compositions of the invention comprise a hyaluronic acidcomponent crosslinked with a crosslinking component, an additive otherthan the crosslinking component; the composition exhibiting reducedTyndall effect when administered into a dermal region of a patient,relative to composition that is substantially identical except withoutthe additive. The composition may be substantially opticallytransparent.

In one embodiment, the additive is a vitamin C derivative, for example,AA2G which may be chemically conjugated to the hyaluronic acid asdescribed elsewhere herein.

In some embodiments, the crosslinking component is BDDE and the degreeof conjugation is between about 3 mol % and about 10 mol %, or up to 15mol % or greater. In some embodiments, the composition further comprisesan anesthetic agent, for example, lidocaine in an amount suitable forproviding comfort to the patient upon injection.

Methods of treating fine lines in the skin of a patient are alsoprovided. The methods generally comprise the steps of introducing intoskin of a patient, a composition such as described herein. For examplethe compositions comprise a mixture of a hyaluronic acid component, acrosslinking component crosslinking the hyaluronic acid, and an additiveother than the crosslinking component, the composition beingsubstantially optically transparent; and wherein the dermal fillercomposition exhibits reduced Tyndall effect relative to composition thatis substantially identical except without the additive.

In some embodiments of the invention, the composition comprises ahyaluronic acid component crosslinked with di- or multiamine crosslinkerusing EDC chemistry. For example, the crosslinker may be HMDA.

In certain embodiments of the invention in which the crosslinker isHMDA, the composition has a G′ of up to about 70 Pa, G″/G′ between about0.65 and about 0.75, extrusion force of about 24 N or less, and a finalHA concentration of between about 24 mg/g and about 25 mg/g.

In certain embodiments of the invention in which the additive is HA-AA2Gconjugate or HA-Vitagen conjugate, the conjugation degree is betweenabout 3 mol % and about 10 mol %, or up to about 15 mol %, or up toabout 40 mol %. These compositions may have a G′ from at least about 30Pa, more preferably at least about 40 Pa, to about 100 Pa, G″/G′ betweenabout 0.30 and about 0.50, extrusion force of about 27 N or less and afinal HA concentration of between about 24 mg/g and about 25 mg/g.

For purposes of the present disclosure, “degree of conjugation” as usedherein is defined as molar percentage of conjugant, e.g., AA2G, to therepeating unit of hyaluronic acid (e.g., HA dimer). Thus, 10 mol %conjugation degree means every 100 HA repeat units contain 10 conjugatedAA2G. Degree of conjugation can be calculated using the methodillustrated in Example 2 below, or other methods known to those of skillin the art.

EXAMPLES Example 1: AA2G Conjugation to Crosslinked HA Gels using BDDEas a Crosslinker

400.6 mg of low molecular weight hyaluronic acid (LMW HA) was hydratedin 1802 mg of 1 wt % NaOH in a syringe for ˜30 min. 800.7 mg of AA2G wasput in a vial, followed by 713.7 mg of BDDE and 1416.8 mg of 10% NaOH.The above solution (pH >12) was allowed to react in a 50° C. water bathfor ˜20 min, before adding to the hydrated HA. After the addition, themixture was mixed ˜20 times by passing back and forth between 2syringes. The mixed paste was put in a vial and in the 50° C. water bathfor ˜2.5 hours. 223.5 mg of 12M HCl was added to 9.05 g PBS, pH7.4.After ˜2.5 hours, the HA-AA2G gel was formed. The gel was cut intopieces, and the HCl-PBS solution was added to it. The gel was allowed toneutralize and swell overnight on an orbital shaker. The gel was sizedthrough a ˜60 μm screen and mixed ˜20 times by passing back and forthbetween 2 syringes. The gel was put in a 15,000 MWCO RC dialysis bag anddialyzed in PBS, pH7.4 buffer. The dialysis went on for ˜185 hours, withfrequent change of PBS buffer. After the dialysis, the gel was put in asyringe and stored in a 4° C. refrigerator.

Example 2: Determination of AA2G Conjugations

The weight of gel as described in Example 1 was noted right beforedialysis, and after dialysis. The assumption was made that the gel was˜1g/mL after dialysis. The dialysis was stopped at the point where nonotable AA2G was coming out per >8 hours in 1 L of PBS. The AA2G wasmeasured at 260 nm using UV/Vis spectrophotometer (Nanodrop 2000C,ThermoScientific). The calibration curve of AA2G was calculated usingdifferent concentration of AA2G in 2% HA (A@260 nm=1.4838 [AA2G(mM)]).

The weight of HA after dialysis: the starting weight of HA×(actualweight before dialysis/theoretical weight)

The mmol of AA2G after dialysis: put the absorption @ 260 nm afterdialysis in the equation (A@260 nm=1.4838 [AA2G(mM)]).

The conjugation @ of AA2G: (mmol of AA2G/mmol of HA)×100%

The AA2G conjugation degree in the gel as described in Example 1 is 14.7mol %.

Example 3: Determination of Gel Rheological Properties

An oscillatory parallel plate rheometer (Anton Paar, Physica MCR 301)was used to measure the properties of the gel obtained in Example 1. Thediameter of plate used was 25 mm. The gap between the plates was set at1 mm. For each measurement, a frequency sweep at a constant strain wasrun first, before the strain sweep at a fixed frequency. The G′ (storagemodulus) was obtained from the strain sweep curve at 1% strain. Thevalue is 1450 Pa for the gel.

Example 4: AA2G Conjugation to Crosslinked HA Gels using BDDE as aCrosslinker, with Tunable Conjugation Degree and Gel TheologicalProperties

The procedure was similar to that as described in Example 1. Conjugationdegree is modified by tuning crosslinker to HA and AA2G mol ratios. Gelproperties were measured as described in Example 3. Details are asfollows:

400.8 mg of LMW HA was hydrated in 1752.1 mg of 1% NaOH in a syringe for˜30 min. 800.3 mg of AA2G was put in a vial, followed by 354.1 mg ofBDDE and 1402.0 mg of 10% NaOH. The above solution (pH >12) was allowedto react in a 50° C. water bath for ˜20 min, before adding to thehydrated HA. After the addition, the mixture was mixed ˜20 times bypassing back and forth between 2 syringes. The mixed paste was put in avial and in the 50° C. water bath for ˜2.5 hours. 140.9 mg of 12M HClwas added to 9.0053 g PBS, pH7.4. After ˜2.5 hours, the HA-AA2G gel wasformed. The gel was cut into pieces, and the HCl-PBS solution was addedto it. The gel was allowed to neutralize and swell overnight on anorbital shaker. The gel was sized through a ˜60 μm screen and mixed ˜20times by passing back and forth between 2 syringes. The gel was put in a15,000 MWCO RC dialysis bag and dialyzed in PBS, pH7.4 buffer. Thedialysis went on for ˜164.5 hours, with frequent change of PBS buffer.After the dialysis, the gel was put in a syringe and stored in a 4° C.refrigerator. The conjugation degree is 13%. Gel storage modulus (G′) is803 Pa.

Example 5: AA2G Conjugation to Crosslinked HA Gels using BDDE as aCrosslinker, Conjugation Degree is 5.3%, G′ is ˜300 Pa

400.3 mg of LMW HA was hydrated in 3002.0 mg of 1% NaOH in a syringe for˜30 min. 800.5 mg of AA2G was put in a vial, followed by 264.3 mg ofBDDE and 1100.0 mg of 10% NaOH. The above solution (pH >12) was allowedto react in a 50° C. water bath for ˜20 min, before adding to thehydrated HA. After the addition, the mixture was mixed ˜20 times bypassing back and forth between 2 syringes. The mixed paste was put in avial and in the 50° C. water bath for ˜2.5 hours. 104.2 mg of 12M HClwas added to 8.5128 g PBS, pH7.4. After ˜2.5 hours, the HA-AA2G gel wasformed, and the HCl-PBS solution was added to it. The gel was allowed toneutralize and swell over the weekend (˜55 hours) on an orbital shaker.The gel was sized through a ˜60 μm screen and mixed ˜20 times by passingback and forth between 2 syringes. The gel was put in a 15,000 MWCO RCdialysis bag and dialyzed in PBS, pH7.4 buffer. The dialysis went on for˜114 hours, with frequent change of PBS buffer. After the dialysis, thegel was put in a syringe and stored in a 4° C. refrigerator. Theconjugation degree and gel rheological properties are measured in aprocedure as described in Example 2 and 3. The conjugation degree is5.3%. Gel storage modulus is ˜300 Pa.

Example 6: AA2G Conjugation to Crosslinked HA Gels using Star-PEGEpoxide as a Crosslinker, Conjugation Degree is 29.4%, G′ is ˜235 Pa

200.4 mg of LMW HA was hydrated in 2000 mg of 1% NaOH in a syringe for˜30 min. 400 mg of AA2G was put in a vial, followed by 312.7 mg ofstar-PEG epoxide and 1026.5 mg of 10% NaOH. The above solution wasallowed to react in a 50° C. water bath for ˜20 min, before adding tothe hydrated HA. After the addition, the mixture was mixed ˜20 times bypassing back and forth between 2 syringes. The mixed paste was put in avial and in the 50° C. water bath for ˜2.5 hours. 187.4 mg of 12M HClwas added to 3.034 g PBS, pH7.4. After ˜2.5 hours, the HA-AA2G gel wasformed, and the HCl-PBS solution was added to it. The gel was allowed toneutralize and swell over the weekend (˜68 hours) on an orbital shaker.The gel was sized through a ˜60 μm screen and mixed ˜20 times by passingback and forth between 2 syringes. The gel was put in a 15,000 MWCO RCdialysis bag and dialyzed in PBS, pH 7.4 buffer. The dialysis went onfor ˜95 hours, with frequent change of PBS buffer. After the dialysis,the gel was put in a syringe and stored in a 4° C. refrigerator. Theconjugation degree and gel rheological properties are measured in aprocedure as described in Examples 2 and 3. The conjugation degree is29.4%. Gel storage modulus is ˜235 Pa.

Example 7: AA2G Conjugation to Crosslinked HA Gels Using Star-PEGEpoxide is a Crosslinker, Conjugation Degree is 27.8%, G′ is ˜363 Pa

200.3 mg of LMW HA was hydrated in 2000 mg of 1% NaOH in a syringe for˜30 min. 400.2 mg of AA2G was put in a vial, followed by 313.4 mg ofstar-PEG epoxide and 1022.6 mg of 10% NaOH. The above solution was addedto the hydrated HA. After the addition, the mixture was mixed ˜20 timesby passing back and forth between 2 syringes. The mixed paste was put ina vial and in the 50° C. water bath for ˜2.5 hours. 196.5 mg of 12M HClwas added to 3.016 g PBS, pH7.4. After ˜2.5 hours, the HA-AA2G gel wasformed, and the HCl-PBS solution was added to it. The gel was allowed toneutralize and swell overnight (˜24 hours) on an orbital shaker. The gelwas sized through a ˜60 μm screen and mixed ˜20 times by passing backand forth between 2 syringes. The gel was put in a 15,000 MWCO RCdialysis bag and dialyzed in PBS, pH7.4 buffer. The dialysis went on for˜98.5 hours, with frequent change of PBS buffer. After the dialysis, thegel was put in a syringe and stored in a 4° C. refrigerator. Theconjugation degree and gel rheological properties are measured in aprocedure as described in Examples 2 and 3. The conjugation degree is27.8%. Gel storage modulus is ˜363 Pa.

Example 8: AA2G Conjugation to Crosslinked HMW HA Gels using BDDE as aCrosslinker, Conjugation Ddegree is about 10 mol %, G′ is about 240 Pa

400.3 mg of HMW HA was hydrated in 2501.3 mg of 4 wt % NaOH in a syringefor ˜30 min. 1200 mg of AA2G was put in a vial, followed by 304.7 mg ofBDDE and 1178.6 mg of 16 wt % NaOH. The above solution (pH >12) wasallowed to react in a 50° C. water bath for ˜20 min and transferred to a20 cc syringe, before adding to the hydrated HA. After the addition, themixture was mixed -20 times by passing back and forth between 2syringes. The mixed paste was put in a 20 cc vial and in the 50° C.water bath for ˜2.5 hours. After ˜2.5 hours, the HA-AA2G gel was formed.Then 226.6 mg of 12M HCl was added to 8492.2 mg 10× PBS, pH7.4 to getHCl-PBS solution and the HCl-PBS solution was added to neutralize andswell the gel. The gel was allowed to neutralize and swell over 48 hrson an orbital shaker. The gel was sized through a ˜60 μm screen andmixed ˜20 times by passing back and forth between 2 syringes. The gelwas put in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH7.4buffer. The dialysis went on for ˜114 hours, with frequent change of PBSbuffer. After the dialysis, the gel was put in a syringe and stored in a4° C. refrigerator. The conjugation degree and gel rheologicalproperties are measured in a procedure as described in Examples, 2 and3. The conjugation degree is 10 mol %. Gel storage modulus is about 240Pa.

Example 9: Vitagen Conjugation to Crosslinked LMW HA Gels using BDDE asa Crosslinker, Conjugation Degree is 15 mol %, G′ is about 365 Pa

398.2 mg of LMW HA was hydrated in 1753.24 mg of 1 wt % NaOH in asyringe for ˜40 min. BDDE (311.7 mg) was added to swollen HA andcontinue let HA swell for another 80 min. The swollen HA/BDDE mixturewas pre-reacted at 50° C. for 20 min.

801.9 mg of Vitagen was separately dissolved in 1459.7 mg of 10 wt %NaOH and mixed with HA which was pre-reacted with BDDE. The mixture wascontinued to react at 50° C. for another 2.5 hrs. After ˜2.5 hours, theHA-Vitagen gel was formed. Then 195 mg of 12M HCl was added to 9004.0 mgof 10× PBS, pH7.4 to get HCl-PBS solution and the HCl-PBS solution wasadded to neutralize and swell the gel. The gel was allowed to neutralizeand swell over 48 hrs on an orbital shaker. The gel was sized through a˜60 μm screen and mixed ˜20 times by passing back and forth between 2syringes. The gel was put in a 20,000 MWCO CE dialysis bag and dialyzedin PBS, pH7.4 buffer. The dialysis went on for ˜120 hours, with frequentchange of PBS buffer. After the dialysis, the gel was put in a syringeand stored in a 4° C. refrigerator. The gel rheological properties weremeasured in a procedure as described in Example 3. The conjugationdegree was determined to be about 15 mol % using a similar method as theAA2G determination as described in Example 2. Gel storage modulus isabout 365 Pa.

Example 10: Vitagen Conjugation to Linear HA Via Amidization Chemistry

200.3 mg of HMW HA was hydrated in 10 ml of water in 60 cc syringe. 500mg of Vitagen was dissolved in 0.5 ml of water and solution wasneutralized to pH 4.8. 197.7 mg of EDC and 149 mg of NHS were dissolvedseparately in 6 ml of water. The above solutions (solutions and EDC/NHSsolutions) are added to another 60 cc syringe containing 23.5 ml ofwater. The two syringes are mixed 20 times by passing back and forthbetween 2 syringes. The mixtures was stored in one syringe and soaked in37° C. bath for 4 hrs. The solutions was finally dialyzed against PBSpH7.4 buffer until no noticeable Vitagen was observed. The conjugationdegree was determined by a similar method as described Example 3. Theconjugation degree is about 10 mol %.

Example 11: AA2P Conjugations to Crosslinked HA Gels

200.4 mg of LMW HA is hydrated in 1000 mg of MES 5.2 buffer in a syringefor ˜30 min. 292 mg of AA2P is put in a vial, followed by 300 mg ofstar-PEG amine added. The above solution is allowed to react at roomtemperature overnight. The gel was hydrated with PBS buffer and dialyzedagainst PBS buffer to remove unreacted AA2P. The finally gel wascharacterized as described in Examples 2 and 3 to determine theconjugation degree and gel rheological properties. The conjugationdegree is about 20 mol %. The storage modulus (G′) is about 500 Pa.

Example 12: Formulation of a HA/BDDE Dermal Filler Product with AA2G forReducing Appearance of Fine Lines

To any of the gels described in the above Examples, after dialysis, asuitable amount of free HA gel may be added to the gel to improve ofmodify gel cohesivity and/or injectability. For example, free HA fibersare swollen in a phosphate buffer solution, in order to obtain ahomogeneous viscoelastic gel (“free” HA gel). This uncrosslinked gel isadded, before the dialysis step, to the HA/BDDE crosslinked gel obtainedin Example 1 (for example, to obtain a composition having between about1% to about 5%, w/w free HA). The resulting gel is then filled intoReady-to-Fill sterile syringes and autoclaved at sufficient temperaturesand pressures for sterilization for at least about 1 minute. Afterautoclaving, the final HA/AA2G product is packaged and distributed tophysicians to use as a dermal filler for superficial injection toimprove the appearance of fine lines in the periorbital or other facialregion.

Example 13: Formulation of HA-AA2G Dermal Filler including Lidocaine

The procedure of Example 12 is followed, but after the dialysis step andbefore the addition of free HA gel, lidocaine chlorhydrate (lidocaineHCl) is added to the mixture. The (lidocaine HCl) in powder form mayfirst be solubilized in WFI and filtered through a 0.2 μm filter. DiluteNaOH solution is added to the cohesive HA/AA2G gel in order to reach aslightly basic pH (for example, a pH of between about 7.5 and about 8).The lidocaine HCl solution is then added to the slightly basic gel toreach a final desired concentration, for example, a concentration ofabout 0.3% (w/w). The resulting pH of the HA/AA2G/lidocaine mixture isthen about 7 and the HA concentration is about 24 mg/g. Mechanicalmixing is performed in order to obtain a proper homogeneity in astandard reactor equipped with an appropriate blender mechanism.

Example 14: Conjugations of Additives Containing Carboxyl FunctionalGroup to HA Hydrogels

Additives such as retinoic acid (AKA, tretinoin), adapalence andalpha-lipoic acid contain carboxyl functional group (—COOH). Theseadditives are conjugated to HA hydrogels via esterifications using EDCchemistry. An example for the conjugations in accordance with anembodiment of the invention is described as follows:

200 mg of HMW HA is hydrated in 10 ml of pH 4.8 MES buffer in 60 ccsyringe. In another syringe, 200 mg of retinoic acid is dissolved in 5ml of water-acetone mixture (water/acetone volume ratio 1:3). The abovetwo syringes are mixed via a syringe connector for about 20 times. Then197.7 mg of EDC and 149 mg of NHS are dissolved separately in 6 ml ofwater in a separate syringe. The syringe containing EDC and NHS isconnected the syringe containing with HA and retinoic acid to allowreactants to mix at least for 20 times by passing back and forth between2 syringes. The mixtures are stored in one syringe and soaked in 37° C.bath for 4 hrs. The gels are dialyzed against isopropanol to removeunconjugated Retinoic acid, and then dialyzed against PBS buffer underaseptic conditions. The gels are packaged into sterilized syringes andstored at 4° C.

Example 15: Conjugations of Additives Containing Hydroxyl FunctionalGroup to HA Hydrogels

Additives such as retinol (AKA, tretinoin), catalase,dimethylaminoethanol and g-Tocopherol contain hydroxyl functional group(—OH). These additives are conjugated to HA hydrogels viaesterifications using EDC chemistry. A typical example for theconjugations is described as follows:

200 mg of HMW HA is hydrated in 10 ml of pH 4.8 2-(N-morpholino)ethanesulfonic acid (MES) buffer in 60 cc syringe. In another syringe,200 mg of retinol acid is dissolved in 5 ml of water-acetone mixture(water/acetone volume ratio 1:3). The above two syringes are mixed via asyringe connector for about 20 times. Then 197.7 mg of EDC and 149 mg ofNHS are dissolved separately in 6 ml of water in a separate syringe. Thesyringe containing EDC and NHS is connected the syringe containing withHA and retinol to allow reactants to mix at least for 20 times bypassing back and forth between 2 syringes. The mixtures are stored inone syringe and soaked in 37° C. bath for 4 hrs. The gels are dialyzedagainst isopropanol to remove unconjugated retinol, and then dialyzedagainst PBS buffer under aseptic conditions. The gels are packaged intosterilized syringes and stored at 4° C.

Example 16: Conjugations of Additives Containing Hydroxyl GunctionalGroup to HA Hydrogels by Post-Modifications

This is a two-step process.

Step one: A crosslinked HA gel, for example, a commercial HA-baseddermal filler, for example, JUVEDERM®, Allergan, Irvine Calif., orRestylane® Medicis Aesthetics, Inc. is treated with EDC/NHS to activatethe carboxyl group of HA.

Step 2: the activated HA hydrogel is treated with additives containinghydroxyl groups. Additives containing hydroxyl groups are retinol,catalase, dimethylaminoethanol and g-Tocopherol hydroxyl functionalgroup (—OH).

A typical examples for the conjugation of additives to crosslinked HAgels is as follows:

2 gm of Juvederm gel is mixed with 200 gm of EDC and 150 mg of NHS atroom temperature. Then 200mg of retinol in 3 ml of acetone-water mixtureis added. The above mixture is reacted at 37° C. for 4 hrs. The gels aredialyzed against isopropanol to remove unconjugated Retinol, and thendialyzed against PBS buffer under aseptic conditions. The gels arepackaged into sterilized syringes and stored at 4° C.

Example 17: Coniugation of Growth Factors, Peptides, or Elastin to HAHydrogels

Additives such as epidermal growth factor (EGF), transforming growthfactor (TGF) and peptides contain functional amine groups may beconjugated to HA to form beneficial dermal fillers. These additives areconjugated to HA via amidization chemistry. A typical example forconjugating is described as follows:

200.3 mg of HMW HA is hydrated in 10 ml of MES pH 5.4 buffer water. 20mg of EGF in 100 mg of MES solution is added. To above mixture, 197.7 mgof EDC and 149 mg are added. The resulting reaction mixture is allowedto react at 37° C. for 4 hrs. After the reaction completes, the gel isfurther dialyzed against isopropanol and then dialyzed against PBSbuffer under aseptic conditions. The gels are packaged into sterilizedsyringes and stored at 4° C.

The present invention further provides methods of enhancing viability ofgrafted adipose tissue. The methods may generally comprise the steps ofintroducing a composition into the skin of a patient adjacent graftedadipose tissue, the composition being a composition as describedelsewhere herein. For example, the composition may comprise hyaluronicacid and a vitamin C derivative covalently conjugated to the hyaluronicacid, wherein a degree of conjugation is between about 3 mol % and about40 mol %. In other aspects of the invention, methods for treating skininclude the steps of introducing, into skin, a composition comprisingadipose tissue, hyaluronic acid and a vitamin C conjugated to thehyaluronic acid.

Example 18: Conjugation of Growth Factors, Peptides, or Elastin to HAHydrogels

To evaluate the mitogenic effects of vitamin C and its derivatives onhuman adipose tissue derived stem cells (hASCs), hASCs were cultured ontissue culture plastic for 4 days in complete MesenPro medium(Invitrogen, Carlsbad, Calif.) supplemented with or without vitamin C(ascorbic acid) or its derivatives (Vitagen or AA2G) in free form.Proliferation was assessed by MTT assay as described by the manufacturer(ATCC, Manassas, VA). After 4 days, concentrations of ascorbic acid,0.25, 0.5, and 1mM, were found to enhance proliferation (measured byamount of conversion of yellow tetrazolium MTT into purple formazan bydehydrogenase enzymes (the purple formazon is solubilized by detergent)by 60%, 80%, and 96% above controls lacking ascorbic acid, respectively.Using the same concentrations of AA2G yielded proliferation enhancementsof 70%, 60%, and 50% above controls, respectively. Similar results wereobtained with Vitagen, showing 70%, 60%, and 30% increases overcontrols, respectively. In summary, vitamin C and its derivatives, AA2Gand vitagen, in the presence of growth factor containing media, enhancehASC proliferation in cell culture.

Crosslinked HA Gels with Conjugated Vitamin C

Preparation of crosslinked HA-based gels with conjugated vitamin C andusing 1,4-butaediol diglycidylether (BDDE) as a crosslinker, inaccordance with certain embodiments of the invention which exhibitreduced Tyndall effect and other advantages are described in Examples 19and 20 below. In Example 19, the vitamin C derivative is ascorbic acid2-glucoside (AA2G) and in Example 20, the vitamin C derivative isascorbyl 3-aminopropyl phosphate (Vitagen). These gels have optimalrheological properties, excellent injectability and high HAconcentration (25 mg/g). Although not wishing to be bound by anyspecific theory of operation, it has been discovered by the presentinventors that crosslinking HA with BDDE in the presence of either AA2Gor Vitagen greatly changes the properties of the gels, with gels havinghigh crosslinked densities, high HA concentrations, low viscosities andlow extrusion forces, relative to commercial HA gels crosslinked withBDDE. Since AA2G or Vitagen is present during crosslinking, the presentgels formed have these ascorbic acid derivatives coupled to the HAchains as both pendent groups, and as crosslinkers bridging HA chains,either alone, or via BDDE. The microscopic structure of the gel isgreatly changed, resulting in gels that have very low extrusion force,even through needles as fine as 30 gauge. Moreover, the gels havebetween about 3 mol % to about 10 mol %, or up to about 15 mol %, ofvitamin C conjugated to HA. When the gels are injected, they releaseactive Vitamin C by endogeneous enzymes such as α-glucosidase fromfibroblasts or phosphotase. The active vitamin C may trigger skincollagenesis and may act as a radical scavenger to inhibit geldegradation.

Example 19: Formulation of a HA/AA2G Gel with reduced Tyndall Effect

A mixture of 400.1 mg of LMW HA and 402.3 mg AA2G in a syringe, washydrated for 60 min after adding 1764.0 mg of a 5 wt % NaOH solution. Ina separate vial was added 800.8 mg of AA2G, followed by 1401.1 mg of an9.1 wt % NaOH solution, and 252.6 mg of BDDE. The resulting solution(pH >12) was allowed to react in a 50° C. water bath for ˜20 min, beforeit was transferred to the hydrated HA. After the addition, the mixturewas mixed ˜20 times by passing it back and forth two syringes. The pastewas then transferred in a vial before it was placed in a 50° C. waterbath for ˜2.5 hours. After crosslinking, a solution containing 197.0 mgof 12 M HCl and 9.18 g 10× PBS, pH 7.4 was added to neutralize the base,and swell the gel for 72 h on an orbital shaker. The gel was sized byforcing it through a ˜60 μm pore size mesh. The sized gel was mixed ˜20times by passing it back and forth two syringes before it wastransferred into a cellulose ester dialysis bag, MWCO ˜20 kDa anddialyzed against PBS, pH 7.4 buffer for 5 days changing the buffer twicedaily. After dialysis, the gel was dispensed into 1 ml COC syringes,centrifuge at 5000 RPM for 5 min to remove air bubbles, and sterilizedwith moist steam. The gel had final HA concentration of 25 mg/g, an AA2Gmol % calculated as described in Example 2 of about 10 mol %, and a G′of about 80 Pa. Other gels were made in a similar manner with G′ valuesof about 60 Pa to about 80 Pa.

Example 19A: Formulation of a HA/AA2G with Lidocaine Gel with ReducedTyndall Effect

To the gel of Example 19, an amount of lidocaine was added to produce aHA/AA2G with lidocaine gel having 0.3% ww lidocaine. A solution oflidocaine was prepared by dissolving lidocaine HCl in PBS buffer pH˜7.4. An aliquot of the lidocaine solution was added to the gel inExample 19 after dialysis but before sterilization. The gel was thenthoroughly mixed to obtain a homogenized mixture with a 0.3% w/wlidocaine concentration.

Example 20: Formulation of a HA/Vitagen Gel with Reduced Tyndall Effect

401.0 mg of LMW HA was hydrated in 2355.0 mg of 1 wt % NaOH solution ina syringe for ˜45 min. 303.8 mg BDDE of was added to the hydrated HA andmix 10 times by syringe-to-syringe mixing. The mixture was pre-reactedin a 50° C. water bath for 15 min. 800.1 mg of Vitagen was separatelydissolved in 950.6 mg of 15 wt % NaOH, followed by 510.1 Milli-Q water.The Vitagen solution was mixed with the pre-heated hydrated HA/BDDEmixture 30 times back and forth using syringe-to-syringe mixing. Themixture was placed back in the 50° C. water bath and the reactionproceeded for another 2 h after which a solution containing 148.1 mg of12M HCl and 8523.1 mg of 10× PBS, pH7.4 was added to the cross TheHCl-PBS solution was added to neutralize and swell the gel. The gel wasallowed to neutralize and swell over 48 hrs on an orbital shaker. Thegel was sized through a ˜60 μm screen and mixed ˜20 times by passingback and forth between 2 syringes. The gel was put in a 20,000 MWCO CEdialysis bag and dialyzed in PBS, pH7.4 buffer. The dialysis went on for˜197 hours, with frequent change of PBS buffer. After the dialysis, thegel was transferred into 1 ml COC syringes, centrifuge at 5000RPM for 5min, and sterilized with moist steam. The final HA concentration of thegel was 24 mg/g.

Crosslinked HA Gels via 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) Chemistry

Preparation of crosslinked HA-based gels, in accordance with certainembodiments of the invention which exhibit reduced Tyndall effect andother advantages are described in Examples 21 and 22 below. In Example21, the gel is made via EDC chemistry using crosslinker is hexamethylenediamine (HMDA), and in Example 20, 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine (4 armamine-4 AA). Crosslinking is carried out under mild conditions, e.g.room temperature, and for example, at pH 5.4. The reactions conditionscould be tuned to prepare highly reticulate gels with optimal gelproperties, excellent injectability and high final HA concentrations(˜24 mg/g). It has been discovered by the inventors that it may beadvantageous to crosslink HA at very low hydration or reactionconcentrations, with a moderate amount of either HMDA or 4 AA, inconjunction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) or sulfonyl-NHS(sulfo-NHS), the coupling agents. The gels will have crosslinked pointsthat are far from each other hence highly crosslinked materials whichhave high damping force. In contrast, crosslinking of HA with BDDE atsuch low hydration or reaction concentrations may be impracticablebecause of the relative inefficiency of the crosslinker.

Example 21: Formulation of a HA/HMDA Gel with Reduced Tyndall Effect

20.0 g of 100 mM MES buffer pH 5.2 was added to a syringe containing1000.0 mg of LMW HA. HMDA solution was prepared by dissolving 260.9 mgHMDA.HCl in 2010.5 mg of 100 mM MES buffer pH 5.2, and adding 2 μl of 1M NaOH to bring pH to 5.2. EDC solution was prepared by dissolving 254.2mg of EDC in 1188.4 mg 100 mM MES buffer pH 5.2, and in a separate vial,44.3 mg of NHS was dissolved in 1341.8 mg of 100 mM MES buffer pH 5.2.Upon full hydration of the HA, ˜1 h, 790 μl of the HMDA solution wasadded to the hydrated HA. The mixture was homogenized by 10 timessyringe-to-syringe mixing. 490 μl EDC and 490 μl NHS solutions were thenadded to the homogenized paste and again mix 10 times bysyringe-to-syringe mixing. The mixture was then transferred to a vialand crosslinked at room temperature for 5 h. before the addition of 17.9ml of 1× PBS buffer pH 7.4. The gel was allowed to swell for 3 days on aroller before it was force through a 60 μm pore size mesh. The sized gelwas placed in a cellulose ester membrane dialysis tubing MWCO 20 KDa anddialyzed against 1× PBS for 4 days changing the buffer twice a day. Thegel was dispensed in 1 ml COC syringes, centrifuge at 5000 RPM for 5min, and sterilized with moist steam. The final HA concentration of thegel was 25 mg/g.

Example 22: Formulation of a HA/4 AA gel with Reduced Tyndall Effect

32.55 g of 100 mM MES buffer pH 5.2 was added to a syringe containing1000.4 mg of LMW HA. 4 AA solution was prepared by dissolving 256.3 mg 4AA in 1039.8 mg of 100 mM MES buffer pH 5.2, and adding 380 μl of 6 MHCl to bring pH to 5.2. EDC solution was prepared by dissolving 251.2 mgof EDC in 1013.8 mg 100 mM MES buffer pH 5.2, and in a separate vial,74.7 mg of NHS was dissolved in 2020.0 mg of 100 mM MES buffer pH 5.2.Upon full hydration of the HA, ˜1 h, 260 μl of the 4 AA solution wasadded to the hydrated HA. The mixture was homogenized by 10 timessyringe-to-syringe mixing. 277 μl EDC and 273 μl NHS solutions were thenadded to the homogenized paste and again mix 10 times bysyringe-to-syringe mixing. The mixture was then transferred to a vialand crosslinked at room temperature for 5 h. before the addition of 6.4ml of 10× PBS buffer pH 7.4. The gel was allowed to swell for 3 days ona roller before it was force through a 60 μm pore size mesh. The sizedgel was placed in a cellulose ester membrane dialysis tubing MWCO 20 KDaand dialyzed against 1× PBS for 4 days changing the buffer twice a day.The gel was dispensed in 1 ml COC syringes, centrifuge at 5000 RPM for 5min, and sterilized with moist steam. The gel had a final HAconcentration of 23 mg/g.

Example 23: Determination of Rheological Properties of Gels of Examples19-22

An Oscillatory parallel plate rheometer, Anton Paar Physica MCR 301, wasused to measure the rheological properties of the gels. A plate diameterof 25 mm was used at a gap height of 1 mm. Measurements were done at aconstant temperature of 25° C. Each measurement consisted of a frequencysweep from 1 to 10 Hz at a constant strain of 2% and a logarithmicincrease of frequency followed by a strain sweep from 1 to 300% at aconstant frequency of 5 Hz with a logarithmic increase in strain. Thestorage modulus (G′) and the viscose modulus (G″) were obtained from thestrain sweep at 1% strain.

Storage and Viscous Moduli of Gels Obtained from Examples 19-22

Sample ID Storage Modulus (G′) Pa Viscous Modulus (G″) Pa Example 19 8425 Example 20 83 33.7 Example 21 67 42 Example 22 41 29.5

Example 24: Extrusion Force Measurements of Gels of Examples 19-22

The force required to extrude the gels through a 30 gauge needle wasmeasured using an Instron 5564 and a Bluehill 2 software. The gels wereextruded from a 1 ml COC syringe through a 30G½ TSK needle. The plungerwas pushed at a speed of 100 mm/min for 11.35 mm, and the extrusionforce was recorded.

Extrusion Force of Gels Obtained from Examples 19-22

Sample ID Extrusion force (N) Example 19 25 Example 20 24 Example 21 22Example 22 19.5

Example 25: Biocompatibility Testing of gels of Examples 19-22

50 μl bolus injections of gel were implanted intradermally in the dorsalsurface of Sprague Dawley rats. The implants were removed at 1 week andanalyzed by histology with hematoxylin and eosin (H&E) staining, andCD68 staining which is a marker for mononuclear inflammation cells.Three 20× images of CD68 were scored from 0-4 based on the degree ofstaining. These values were then averaged out to give a sample score.Four samples were analyzed from each gel.

Average CD68 Scores of Examples 19-22

Sample ID Score Example 1 1.8 Example 2 1.6 Example 3 2.7 Example 4 1.3

Example 26: Cytotoxicity Testing of Gels of Examples 19-22, ISO 10993-5

In Vitro cytotoxicity tests of the gels were performed by NAMSAaccording to the Agarose Overlay Method of ISO 10993-5: biologicalEvaluation of Medical Devices-Part 5: Tests for In Vitro Cytotoxicity.Triplicate wells were dosed with 0.1 ml of test articles placed on afiltered disc, as well as 0.9% NaCI solution, 1 cm length of highdensity polyethylene as a negative control, and 1×1 cm² portion of latexas a positive control. Each was placed on an agarose surface directlyoverlaying a monolayer of L929 mouse fibroblast cells. After incubatingat 37° C. in 5% CO₂ for 24 h. the cultures were examined macroscopicallyand microscopically for any abnormal cell morphology and cell lysis. Thetest articles were scored from 0-4 based on the zone of lysis in theproximity of the samples. Test materials from examples 1, 3, and 4scored 0 as test articles showed no evidence of causing any cell lysisor toxicity.

Quantitative Analysis of Tyndall Effect

In order to further support visual observations and carry outcomparative performance analysis of HA fillers, it was deemed necessaryto do a quantitative analysis of Tyndall effect. As such no quantitativetechniques for Tyndall effect specific to dermal fillers exist in theliterature. However, based on existing scientific understanding on lightscattering and interaction of light with skin, two distinct approachesbased on (a) colorimetry, and (b) spectroscopy were employed to quantifyTyndall effect in skin. Based on these techniques three distinctquantitative parameters (outlined below) were defined to measure Tyndalleffect in vivo.

-   a) Tyndall Effect Visual Score: The scale had a range of 1 to 5 with    increments of 0.5. A score of 1 was given to injection sites with    normal skin tone and no blue discoloration. A maximum score of 5 was    given to thick and pronounced blue discoloration (typically    associated with Restylane or Juvéderm Ultra Plus). Three independent    observers were trained on the scale before being blinded to score    test samples.-   b) Blue component of skin color—“b”: A chromameter (CM2600D, Konica    Minolta, N.J.) was used to quantify the blue color component of    light remitted from skin sites injected with the various fillers.    This was achieved by using the “b” component of L-a-b color scale.-   c) “% Blue Light” remitted from skin: A portable spectrophotometer    (CM2600D, Konica Minolta, N.J.) was used to quantify the % blue    light remitted from skin in the total visible light range. This was    achieved by integrating the area under the visible light spectrum    between 400-490 nm and normalizing it by the total area under the    spectrum (400-700 nm).

Example 27: Tyndall Evaluation of Gels

Gels were injected intradermally through a 27 G½ TSK needle using linearthreading technique into the thighs of two months old hairless rats. Thegels were implanted superficially to mimic clinical fine lineprocedures. Tests for Tyndall were performed 48 h after gelimplantation. Before performing the Tyndall tests, the animals wereeuthanized to improve contrast of the Tyndall effect due to lack ofhemoglobin.

Images of gels from Examples 19 and 21, 2 days after implantation, areshown in FIG. 12. Images for commercial Juvéderm Refine and RestylaneTouch are also shown for comparison. A bluish line (Tyndall effect) isclearly visible in the images of commercial gels Juvéderm Refine andRestylane Touch . Gels from Examples 19, 19A (not shown) and 21exhibited no Tyndall effect.

A visual score of 1-5 with increments of 0.5, was used to score theinjection sites. Injection sites with score of 1 showed no skindiscoloration, while injections sites with score of 5 showed severe bluediscoloration of the skin. Spectroscopic analyses were also performed onthe injection sites with the aid of a chromatometer (CM2600D, KonicaMinolta, N.J.). The blue component of skin color “b”, and the % of bluelight remitted from skin (400-700 nm) were independently measured. FIGS.13 and 14, show visual Tyndall score and % of blue light remitted. Gelsfrom Examples 19 and 21 showed no Tyndall effect, and had lower visualTyndall score and % of blue light remitted values. The Tyndall score and% of remitted blue light values were higher for Juvéderm Refine andRestylane Touch. Belotero Soft did not show any Tyndall and values werecomparable to those of Examples 19 and 21. See FIGS. 13 and 14.

Example 28: In Vivo Duration Evaluation of Gels by Histology

50 μl bolus injections of gels of the invention and commercial gels wereimplanted intradermally in the dorsal surface of Sprague Dawley rats.The implants were removed at 1 week and analyzed by histology withhematoxylin and eosin (H&E) staining. Sections were taken at exactly atthe injection sites. Two sections were cut from each tissue sample andthe H&E stained section was stitched using a stitching scope. Thesamples were then grouped and scored as follows; none (0%), low (25%),medium (50%), and high (100%) depending on the amount of materialremaining. See FIG. 15.

Example 28A: In Vivo Duration Evaluation of Gels by MRI

Magnetic Resonance Imaging (MRI) study was used to evaluate the volumeand surface area change with time of gels of t he invention andcommercial gels over a period of 40 weeks, after intradermal injectionsin female Sprague-Dawley rats. The gels were injected at a target volumeof 150 μl per implant. Implants were located at two contralateral sitesslightly caudal to shoulder, two contralateral sites slightly rostralfrom knee, and two contralateral sites midpoint between head and tail.MRI scans were performed on a 7 Tesla 70/30 Bruker Biospec MRI scanner.Images were collected on the day of implantation (week 0), and at 12,24, 40 weeks after implantation. Plots of absolute volume of gel versusTime is shown in FIG. 16 below. High persistence gels have high absolutevolume at 40 weeks implantation.

Example 29: Compositions of the Invention as Used in the Treatment ofPeriorbital Lines

A 40 year old thin woman presents with fine wrinkles in the periorbitalregion and requests dermal filler treatment. Using a 30 gauge needle,the physician introduces 0.6 ml of a HA-based gel in accordance with theinvention (such as that described in Example 19) superficially into thefine lines beneath each of her eyes and in the tear trough region usinglinear threading technique. Although the gel is introducedsuperficially, no blue discoloration is observed and the patient issatisfied with the results.

As shown, compositions of the present invention, for example, those ofExamples 19 and 21, have reduced or insignificant Tyndall effect, andsubstantially longer duration in the body relative to certain HA-basedcommercial gels, for example, Juvederm Refine/Surgiderm 18 and BeloteroSoft. Relative to Belotero Soft, the gels of the present invention hadnot only a Tyndall score at least as favorable as this commercial gel,but advantageously exhibited substantially higher in vivo duration.

Example 30: Injectable Compositions of the Invention for Improvement ofthe Appearance of Fine Lines

Additives such as Vitamin A, Vitamin B, Vitamin C, Vitamin D, Vitamin Eand derivatives thereof, alone and in combination, are conjugated tocrosslinked hyaluronic acid gels in a manner so as to produce a varietyof substantially optically transparent, injectable HA-based gels. The HAcomponent is at least 90% by weight, for example, is substantiallyentirely low molecular weight HA, or about 100% low molecular weight HA,as defined elsewhere herein. These additives are conjugated to HAhydrogels using any suitable means. The conjugated gels are sized andprocessed to produce an injectable, pH neutral, cohesive compositionhaving a HA concentration of at least about 20 mg/g, for example, about23, about 24 mg/g, about 25 mg/g, up to about 30 mg/g, and suitable forinjection through a fine gauge needle. The gels have a G′ value of atleast about 50 PA, about 60 Pa, about 70 Pa, about 80 Pa up to, and nogreater than about 100 Pa. The gels are packaged and sterilized usingautoclave, UV light or other suitable means.

Each of the gels is useful for superficial injection, for example,injection into skin at a depth of no greater than about 1.0 mm, in awrinkle of patients, for example, the periorbital region, nasolabialfold region, tear trough region, neck region, or any other facial regionthat would benefit from dermal filling. Despite the superficialintroduction of the gels, no discoloration due to Tyndall effect isobserved and the patients are satisfied with the results.

In closing, it is to be understood that although aspects of the presentspecification have been described with reference to the variousembodiments, one skilled in the art will readily appreciate that thespecific examples disclosed are only illustrative of the principles ofthe subject matter disclosed herein. Therefore, it should be understoodthat the disclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,those skilled in the art could make numerous and various modificationsor changes to or alternative configurations of the disclosed subjectmatter can be made in accordance with the teachings herein withoutdeparting from the spirit of the present specification. Changes indetail may be made without departing from the spirit of the invention asdefined in the appended claims. Lastly, the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. In addition, it is intended that all mattercontained in the above description or shown in the accompanying drawingsshall be interpreted as illustrative only and not limiting. Accordingly,the present invention is not limited to that precisely as shown anddescribed.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used herein,the term “about” means that the item, parameter or term so qualifiedencompasses a range of plus or minus ten percent above and below thevalue of the stated item, parameter or term. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thespecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents arebased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A dermal filler composition comprising hyaluronicacid and a vitamin C derivative covalently conjugated to the hyaluronicacid.
 2. The dermal filler composition of claim 1, wherein the vitamin Cderivative is conjugated with the hyaluronic acid with a degree ofconjugation of between about 3 mol % and about 15 mol %.
 3. The dermalfiller composition of claim 1, wherein the vitamin C derivative isconverted into vitamin C in vivo by enzymatic cleavage.
 4. The dermalfiller composition of claim 1, wherein the vitamin C derivative isselected from ascorbic acid 2-glucoside (AA2G), ascorbyl 3 aminopropylphosphate and sodium ascorbyl phosphate (AA2P).
 5. The dermal fillercomposition of claim 4, wherein the vitamin C derivative is ascorbicacid 2-glucoside (AA2G).
 6. The dermal filler composition of claim 4,wherein the vitamin C derivative is ascorbyl 3-am inopropyl phosphate.7. The dermal filler composition of claim 4, wherein the vitamin Cderivative is sodium ascorbyl phosphate (AA2P).
 8. The dermal fillercomposition of claim 1, wherein the hyaluronic acid is crosslinked. 9.The dermal filler composition of claim 8, wherein the hyaluronic acid iscrosslinked with 1,4-butanediol diglycidyl ether (BDDE), pentaerythritolglycidal ether (Star-PEG epoxide), pentaerythritol (3-aminopropyl) ether(Star-PEG amine), or lysine.
 10. The dermal filler composition of claim9, wherein the hyaluronic acid is crosslinked with 1,4-butanedioldiglycidyl ether (BDDE).
 11. The dermal filler composition of claim 9,wherein the hyaluronic acid is crosslinked with lysine.
 12. The dermalfiller composition of claim 1, further comprising an anesthetic agent.13. The dermal filler composition of claim 12, wherein the anestheticagent is lidocaine.
 14. A method for treating a soft tissue condition ina subject in need thereof, the method comprising: administering a dermalfiller composition comprising hyaluronic acid and a vitamin C derivativecovalently conjugated to the hyaluronic acid.
 15. The method of claim14, wherein the soft tissue condition is a wrinkle or scar.
 16. Themethod of claim 14, wherein the dermal filler composition releases thevitamin C derivative in the subject in need thereof for at least about 1month and up to about 20 months.
 17. The method of claim 14, wherein thevitamin C derivative is covalently conjugated to the hyaluronic acidwith BDDE or lysine.
 18. The method of claim 14, wherein the vitamin Cderivative is selected from ascorbic acid 2-glucoside (AA2G), ascorbyl 3aminopropyl phosphate and sodium ascorbyl phosphate (AA2P).
 19. Themethod of claim 14, wherein the vitamin C derivative is AA2G.
 20. Themethod of claim 14, wherein the vitamin C derivative is AA2P.